Novel major histocompatibility complex (MHC) allele discovery

• from cDNA libraries by Sanger sequencing

• MHC class I

• MHC class II

• from cDNA amplicons by Sanger sequencing

• MHC class I

• MHC class II

• from cDNA amplicons by Roche/454 pyrosequencing

• MHC class I

• MHC class II

Major histocompatibility complex genotyping

• PCR-SSP for 29 common Indian rhesus macaque MHC class I alleles

• PCR-SSP for 8 common Chinese rhesus macaque MHC class I alleles

• PCR-SSP for 4 common Mauritian cynomolgus macaque MHC class I alleles

• Roche/454 pyrosequencing for rhesus, cynomolgus, and pig-tailed macaques

• FLX chemistry

• Titanium chemistry

• DNA microsatellite haplotyping for rhesus, cynomolgus, and pig-tailed macaques

• UW-Madison 16 marker panel

• Biomedical Primate Research Center XX marker panel

Novel major histocompatability complex (MHC) allele discovery from cDNA amplicons by Sanger sequencing – MHC class I

1. Total RNA isolation from whole blood or cells

[MagNA Pure LC RNA Isolation Kit – High Performance (Roche Applied Science), alternatively other RNA isolation protocols may be utilized]

(a) Ensure that the MagNA Pure LC Instrument has the proper RNA purification protocols installed as detailed by the MagNA Pure LC RNA Isolation Kit manual.
(b) Prepare the isolation kit reagents as detailed in the manual.
(c) Aliquot 200 µl of whole blood or white blood cells, or 1 x 106 peripheral blood mononuclear or cultured cells as directed by the manual.
(d) Set up the instrument using the appropriate protocol for the starting material. Follow instructions supplied by the software, as it will calculate reagent volumes based upon number of samples to be isolated.
(e) Immediately upon completion of the isolation run, quantitate RNA samples with a NanoDropTM (Thermo Scientific) prior to proceeding with cDNA synthesis. If necessary, RNA samples can be stored in RNase-free microcentrifuge tubes at -80 °C.

2. Synthesis of first-strand cDNA

[MJ Research Tetrad Thermocycler (Bio-Rad Labortories) and Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen)], alternatively other thermocyclers may be utilized.

(a) Prepare a reaction mixture of 1 µl 10 mM dNTP mix, 1 µl 50 µM Oligo(dT)20, and 50 ng total RNA (or up to 8 µl if 50 ng RNA would exceed 8 µl). If necessary, add DEPC-treated water to obtain a 10 µl final reaction volume.
(b) Incubate reactions at 65 °C for 5 min to denature. During this incubation, prepare the cDNA synthesis master mix of 2 µl 10X RT Buffer, 4 µl 25 mM MgCl2, 2µl 0.1 M DTT, 1 µl RNaseOUTTM, and 1 µl SuperScriptTM III RT.
(c) Incubate initial reaction mixture at 4 °C or on ice for at least 1 min. During this incubation, add 10 µl cDNA synthesis master mix to initial reaction mixture.
(d) Incubate at 50 °C for 50 min.
(e) Terminate cDNA synthesis by incubating at 85 °C for 5 min.
(f) Add 1 µl 2U/µl E. coli RNase H to reaction mixture and incubate at 37 °C for 20 min to remove any remaining RNA.
(g) Upon completion, proceed directly to PCR or store cDNA at -20 °C.

3. PCR amplification of MHC class I cDNA

[PhusionTM High-Fidelity PCR Master Mix with HF Buffer (New England BioLabs), nuclease-free water, Flash- Gel DNA Cassette 1.2% (Lonza), FlashGel_ 5X loading dye (Lonza), FlashGel_ Quant Ladder 100–1.5 kb (Lonza)]

(a) Prepare 50 µl primer-specific reaction mixtures of 25 µl 2X PhusionTM High-Fidelity PCR Master Mix, 20 µl nuclease-free water, 1.25 µl 5 µM 5’ PCR primer, 1.25 µl 5 µM 3’ PCR primer (A or B), and 2.5 µl cDNA.
(b) PCR amplify using the following cycle conditions for the 3’ MHC class IA primer: 98 °C for 3 min, 20–30 cycles of 98 °C for 5 s, 60 °C for 1 s, 72 °C for 20 s, and a final elongation of 72 °C for 5 min. For the 3’ MHC class IB primer, increase the annealing temperature to 63 °C; all other temperatures and times are unchanged.
(c) After 20 cycles, check a 4 µl aliquot of each reaction on a FlashGel DNA cassette (Lonza) following the manufacturer’s protocol. Add additional cycles to reactions as needed to obtain sufficient amplification of each sample.

5’ PCR primer    :    5’MHC-UTR        5’-[MID]AGAGTCTCCTCAGACGCCGAG-3’
3’ IA PCR primer:    3’MHC-UTR-A     5’-CAGGAACAYAGACACATTCAGG-3’
3’ IB PCR primer:    3’MHC-UTR-B     5’-GGCTGTCTCTCCACCTCCTCAC-3’

MID tag set:

4. Agarose gel electrophoresis and amplicon purification

[1X TAE, genetic analysis grade agarose, 6X loading dye, SYBR Safe DNA gel stain 10,000X concentrate in DMSO (Invitrogen), BenchTop 1 kb DNA ladder (Promega), MinElute Gel Extraction Kit (Qiagen)]


(a) Prepare a 1% agarose gel by mixing 150 ml 1X TAE with 1.5 g agarose.
(b) Heat the mixture in the microwave to melt the agarose, swirling occasionally.
(c) Add 15 µl SYBR_ Safe DNA gel stain to the mixture, swirling to mix. Pour gel and allow to solidify.
(d) Add 10 µl 6X loading dye to each PCR reaction and load entire volume into a single well of the agarose gel. Space samples appropriately leaving at least one empty well between samples to avoid cross-contamination. Load 6 µl ladder into an empty well for sizing of the ~1.25 kb amplicon.
(e) Run agarose gel in 1X TAE buffer at 120 V for 1 h. Ensure that the artifactual products have sufficiently separated from the ~1.25 kb amplicon to allow for clean excision. Extend running time if necessary.
(f) Once sufficient separation is observed, visualize with a ‘DNA safe’ blue light transilluminator (Invitrogen) and excise the ~1.25 kb amplicon band. Place the gel slice into a colorless 1.5 ml microcentrifuge tube.
(g) Proceed with gel slice purification following the appropriate MinElute_ protocol. Elute the DNA with 10 µl nuclease-free water. Typical post-elution DNA concentrations range from 2–15 ng/ul.

5. Purified DNA quantitation and pooling

[Quant-iTTM dsDNA HS Assay Kit (Invitrogen), QubitTM assay tubes (Invitrogen)]

(a) Prepare the Quant-iTTM working solution, dilute standards and samples, calibrate the fluorometer, read samples, and calculate concentrations of samples as directed by the manufacturer’s protocol.
(b) If pooling products for ligation, normalize all products for each pool to the concentration of the least concentrated sample. Combine equal volumes of the normalized products to create each pool.
(c) Proceed directly to ligation and transformation or store DNA products at -20 °C.

6. Ligation and transformation of blunt-end PCR amplicons

[Zero Blunt TOPO PCR Cloning Kit with One Shot TOP10 Chemically Competent E. coli (Invitrogen), 50 µg/ml kanamycin stock, 50 µg/ml ampicillin stock, Ready2AUTOCLAVE LB (Miller’s) broth agar powder (ISC BioExpress), 50 µg/ml kanamycin stock, CIRCLEGROW broth powder (Qbiogene)]

(a) Prepare LB agar plates containing 50 µg/ml kanamycin (2 or more per ligation to be performed) and LB agar plates containing 50 µg/ml ampicillin (positive control).
(b) Prepare ligation mixture of 1 µl salt solution (1.2 M NaCl + 0.06 M MgCl2), 1 µl pCR_-BluntII-TOPO_, and 4 µl purified PCR product (single or pooled amplicons; typical concentration range 8–60 ng per ligation reaction). Use nuclease-free water in place of PCR product for a negative control.
(c) Incubate ligation at room temperature for 30 min.
(d) During the last 10 min of the ligation incubation, thaw TOP10 chemically competent cells on ice.
(e) Add 4 µl of incubated ligation mixture to TOP10 cells, stirring with pipette tip to mix (do not pipette up and down). Add 1 µl 10 pg/µl pUC19 control DNA to cells for a positive transformation control.
(f) Incubate ligation mixture with TOP10 cells on ice for 5 min.
(g) Heat shock cells at 42 °C for 30 s.
(h) Recover cells on ice for 2 min.
(i) Add 250 µl room temperature S.O.C. medium and incubate at 37 °C shaking the tubes horizontally at 200 rpm for 1 h.
(j) During the incubation, warm the LB + kanamycin plates (LB + ampicillin plates for the positive control) to 37 °C.
(k) Spread two 150 µl volumes of each transformation mixture onto two LB + kanamycin plates. Spread 150 µl of the positive control onto an LB + ampicillin plate.
(l) Incubate LB selective plates at 37 °C overnight.
(m) Prepare CIRCLEGROW® (or LB) broth plus 50 µg/ml kanamycin.
(n) Pick 48 class IA clones per animal (192 class IA clones for a ligation pool of 4 animals), 144 class IB clones per animal (192–288 class IB clones for a ligation pool of 2 animals) into 1.3 ml selective liquid media in 96-well deep-well culture plates. For a control of the kanamycin-selective liquid media, pick a clone from the positive transformation control plate into the broth + kanamycin in a separate bacterial culture tube (since the positive control colonies are resistant to ampicillin, not kanamycin, there should be no growth in the control).
(o) Incubate deep-well culture plates at 37 °C overnight, shaking at 200 rpm.

7. Plasmid DNA purification and restriction enzyme digestion

[PerfectprepTM Plasmid 96 Vac Bind Kit (5 PRIME), EcoRI and 10X NEBuffer EcoRI (New England BioLabs), nuclease-free water, 1X TAE, genetic analysis grade agarose, SYBR Safe DNA gel stain 10,000X concentrate in DMSO (Invitrogen), 6X loading dye, BenchTop 1 kb DNA ladder (Promega)]

(a) Centrifuge the deep-well culture plates at 1900g for 5 min to pellet bacteria. Pour off supernatant, invert and blot any remaining liquid medium to a paper towel.
(b) Proceed with plasmid DNA purification following manufacturer’s protocol. Typical post-miniprep concentrations range from 50–150 ng/µl.
(c) To perform the EcoRI restriction digest, make a reaction mix of 0.5 µl EcoRI enzyme, 1 µl 10X NEBuffer EcoRI, 5.5 µl nuclease-free water, and 3 µl purified plasmid DNA.
(d) Incubate digestion mixture at 37 °C for 1 h.
(e) During incubation, prepare a 1% agarose gel as described above in 4.4 steps (a–c).
(f) Add 2 µl 6X loading dye to each digest and load into agarose gel. Load 6 µl ladder into an empty well for sizing the ~3.5 kb vector and the ~1.25 kb insert.
(g) Run agarose gel in 1X TAE buffer at 120 V for 15 min. Analyze the gel to calculate the frequency of clones with the desired insert (generally > 85%).

8. Single-pass cycle sequencing of cDNA clones

[5’Refstrand (5’-GCTACGTGGACGACACGC- 3’), SBT190-R (5’-TCGCTCTGGTTGTAGTAGC-3’), DYEnamicTM ET Terminator Cycle Sequencing Kit (GE Healthcare), CleanSEQ_ Dye-Terminator Removal Kit (Agencourt), 85% ethanol (EtOH)]

(a) Prepare a 5 mM MgCl2, 20 mM Tris sequencing buffer by adding 0.102 g MgCl2 and 0.242 g Tris to 80 ml double-distilled water, stir to dissolve, adjust pH to 9.0, and bring volume to 100 ml with double-distilled water.
(b) Prepare sequencing reaction mix containing 1 µl DYEnamicTM ET Terminator, 1 µl 3.2 µM oligo (either 5’Refstrand or SBT190-R), 6 µl sequencing buffer, 7 µl nuclease-free water, and 5 µl purified plasmid DNA.
(c) Cycle sequence using the following thermocycler profile: 30 cycles of 95 °C for 20 s, 50 °C for 15 s, and 60 °C for 1 min.
(d) Purify the sequencing reactions using the Agencourt_ CleanSEQ_ Dye-Terminator Removal kit following the manufacturer’s protocol for ET Terminator clean-up.
(e) Resolve the cleaned sequencing products by capillary electrophoresis on an ABI 3730 Genetic Analyzer (Applied Biosystems), or a comparable Sanger sequencing platform.

9. Sequence analysis

(a) Edit single-pass sequence results for high quality data using CodonCode Aligner (CodonCode Corporation) or similar sequence analysis software. Clip sequence ends to maximize the region with an error rate below 0.01 and delete short sequences with less than 50 Phred 20 quality bases.
(b) Export high quality sequence for BLASTn analysis. Use the following parameters to most effectively analyze MHC class I alleles through the NCBI website: ‘Others (nr etc.)’ database, ‘Nucleotide collection (nr/nt)’, limit organism to the genus and species of NHP being analyzed (such as Macaca mulatta), and optimize for ‘Highly similar sequences (megablast)’. Under ‘Algorithm parameters’, ‘General Parameters’, limiting max target sequences to ‘10’ will also simplify the results.
(c) Determine which cDNA clones are identical to one or more known MHC class I alleles by looking for 100% identity to any alleles listed in GenBank.
(d) Determine the closest match for any sequences lacking 100% identity with any alleles deposited in GenBank.
(e) Perform any additional sequencing reactions necessary to fully characterize these novel alleles as described above in section 4.8, using other previously described MHC class I bidirectional sequencing oligos T7, M13, 3’Refstrand, and 5’Transmembrane
(f) Deposit novel full-length sequences to GenBank and the Immuno Polymorphism Database (IPD) to obtain official allele names.

Novel major histocompatability complex (MHC) allele discovery from cDNA amplicons by Sanger sequencing – MHC class II

1. Total RNA isolation from whole blood or cells

[MagNA Pure LC RNA Isolation Kit – High Performance (Roche Applied Science), alternatively other RNA isolation protocols may be utilized]

(a) Ensure that the MagNA Pure LC Instrument has the proper RNA purification protocols installed as detailed by the MagNA Pure LC RNA Isolation Kit manual.
(b) Prepare the isolation kit reagents as detailed in the manual.
(c) Aliquot 200 µl of whole blood or white blood cells, or 1 x 106 peripheral blood mononuclear or cultured cells as directed by the manual.
(d) Set up the instrument using the appropriate protocol for the starting material. Follow instructions supplied by the software, as it will calculate reagent volumes based upon number of samples to be isolated.
(e) Immediately upon completion of the isolation run, quantitate RNA samples with a NanoDropTM (Thermo Scientific) prior to proceeding with cDNA synthesis. If necessary, RNA samples can be stored in RNase-free microcentrifuge tubes at -80 °C.

2. Synthesis of first-strand cDNA

[MJ Research Tetrad Thermocycler (Bio-Rad Labortories) and Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen)], alternatively other thermocyclers may be utilized.

(a) Prepare a reaction mixture of 1 µl 10 mM dNTP mix, 1 µl 50 µM Oligo(dT)20, and 50 ng total RNA (or up to 8 µl if 50 ng RNA would exceed 8 µl). If necessary, add DEPC-treated water to obtain a 10 µl final reaction volume.
(b) Incubate reactions at 65 °C for 5 min to denature. During this incubation, prepare the cDNA synthesis master mix of 2 µl 10X RT Buffer, 4 µl 25 mM MgCl2, 2µl 0.1 M DTT, 1 µl RNaseOUTTM, and 1 µl SuperScriptTM III RT.
(c) Incubate initial reaction mixture at 4 °C or on ice for at least 1 min. During this incubation, add 10 µl cDNA synthesis master mix to initial reaction mixture.
(d) Incubate at 50 °C for 50 min.
(e) Terminate cDNA synthesis by incubating at 85 °C for 5 min.
(f) Add 1 µl 2U/µl E. coli RNase H to reaction mixture and incubate at 37 °C for 20 min to remove any remaining RNA.
(g) Upon completion, proceed directly to PCR or store cDNA at -20 °C.

3. PCR amplification of MHC class II cDNA

[PhusionTM High-Fidelity PCR Master Mix with HF Buffer (New England BioLabs), nuclease-free water, Flash- Gel DNA Cassette 1.2% (Lonza), FlashGel_ 5X loading dye (Lonza), FlashGel_ Quant Ladder 100–1.5 kb (Lonza)]

(a) Prepare 50 µl primer-specific reaction mixtures of 25 µl 2X PhusionTM High-Fidelity PCR Master Mix, 20 µl nuclease-free water, 1.25 µl 5 µM 5’ PCR primer, 1.25 µl 5 µM 3’ PCR primer, and 2.5 µl cDNA.
(b) PCR amplify using the following cycle conditions: 98 °C for 3 min, 29 cycles of 98 °C for 5 s, 63 °C for 1 s, 72 °C for 20 s, and a final elongation of 72 °C for 5 min.
(c) After 20 cycles, check a 4 µl aliquot of each reaction on a FlashGel DNA cassette (Lonza) following the manufacturer’s protocol. Add additional cycles to reactions as needed to obtain sufficient amplification of each sample.

Primers used to amplify MHC class II alleles:

4. Agarose gel electrophoresis and amplicon purification

[1X TAE, genetic analysis grade agarose, 6X loading dye, SYBR Safe DNA gel stain 10,000X concentrate in DMSO (Invitrogen), BenchTop 1 kb DNA ladder (Promega), MinElute Gel Extraction Kit (Qiagen)


(a) Prepare a 1% agarose gel by mixing 150 ml 1X TAE with 1.5 g agarose.
(b) Heat the mixture in the microwave to melt the agarose, swirling occasionally.
(c) Add 15 µl SYBR_ Safe DNA gel stain to the mixture, swirling to mix. Pour gel and allow to solidify.
(d) Add 10 µl 6X loading dye to each PCR reaction and load entire volume into a single well of the agarose gel. Space samples appropriately leaving at least one empty well between samples to avoid cross-contamination. Load 6 µl ladder into an empty well for sizing of the amplicon.
(e) Run agarose gel in 1X TAE buffer at 120 V for 1 h. Ensure that the artifactual products have sufficiently separated from the amplicon of interest to allow for clean excision. Extend running time if necessary.
(f) Once sufficient separation is observed, visualize with a ‘DNA safe’ blue light transilluminator (Invitrogen) and excise the amplicon band. Place the gel slice into a colorless 1.5 ml microcentrifuge tube.
(g) Proceed with gel slice purification following the appropriate MinElute_ protocol. Elute the DNA with 10 µl nuclease-free water. Typical post-elution DNA concentrations range from 2–15 ng/ul.

5. Purified DNA quantitation

[Quant-iTTM dsDNA HS Assay Kit (Invitrogen), QubitTM assay tubes (Invitrogen)]

(a) Prepare the Quant-iTTM working solution, dilute standards and samples, calibrate the fluorometer, read samples, and calculate concentrations of samples as directed by the manufacturer’s protocol.
(b) Proceed directly to ligation and transformation or store DNA products at -20 °C.

6. Ligation and transformation of blunt-end PCR amplicons

[Zero Blunt TOPO PCR Cloning Kit with One Shot TOP10 Chemically Competent E. coli (Invitrogen), 50 µg/ml kanamycin stock, 50 µg/ml ampicillin stock, Ready2AUTOCLAVE LB (Miller’s) broth agar powder (ISC BioExpress), 50 µg/ml kanamycin stock, CIRCLEGROW broth powder (Qbiogene)]

(a) Prepare LB agar plates containing 50 µg/ml kanamycin (2 or more per ligation to be performed) and LB agar plates containing 50 µg/ml ampicillin (positive control).
(b) Prepare ligation mixture of 1 µl salt solution (1.2 M NaCl + 0.06 M MgCl2), 1 µl pCR-BluntII-TOPO, and 4 µl purified PCR product (single or pooled amplicons; typical concentration range 8–60 ng per ligation reaction). Use nuclease-free water in place of PCR product for a negative control.
(c) Incubate ligation at room temperature for 30 min.
(d) During the last 10 min of the ligation incubation, thaw TOP10 chemically competent cells on ice.
(e) Add 4 µl of incubated ligation mixture to TOP10 cells, stirring with pipette tip to mix (do not pipette up and down). Add 1 µl 10 pg/µl pUC19 control DNA to cells for a positive transformation control.
(f) Incubate ligation mixture with TOP10 cells on ice for 5 min.
(g) Heat shock cells at 42 °C for 30 s.
(h) Recover cells on ice for 2 min.
(i) Add 250 µl room temperature S.O.C. medium and incubate at 37 °C shaking the tubes horizontally at 200 rpm for 1 h.
(j) During the incubation, warm the LB + kanamycin plates (LB + ampicillin plates for the positive control) to 37 °C.
(k) Spread two 150 µl volumes of each transformation mixture onto two LB + kanamycin plates. Spread 150 µl of the positive control onto an LB + ampicillin plate.
(l) Incubate LB selective plates at 37 °C overnight.
(m) Prepare CIRCLEGROW (or LB) broth plus 50 µg/ml kanamycin.
(n) Pick clones and place into 1.4 ml selective liquid media in 96-well deep-well culture plates. For a control of the kanamycin-selective liquid media, pick a clone from the positive transformation control plate into the broth + kanamycin in a separate bacterial culture tube (since the positive control colonies are resistant to ampicillin, not kanamycin, there should be no growth in the control).
(o) Incubate deep-well culture plates at 37 °C for 17 to 24 h, shaking at 200 rpm.

7. Plasmid DNA purification and restriction enzyme digestion

[PerfectprepTM Plasmid 96 Vac Bind Kit (5 PRIME), EcoRI and 10X NEBuffer EcoRI (New England BioLabs), nuclease-free water, 1X TAE, genetic analysis grade agarose, SYBR Safe DNA gel stain 10,000X concentrate in DMSO (Invitrogen), 6X loading dye, BenchTop 1 kb DNA ladder (Promega)]

(a) Centrifuge the deep-well culture plates at 1900g for 5 min to pellet bacteria. Pour off supernatant, invert and blot any remaining liquid medium to a paper towel.
(b) Proceed with plasmid DNA purification following manufacturer’s protocol. Typical post-miniprep concentrations range from 50–150 ng/µl.
(c) To perform the EcoRI restriction digest, make a reaction mix of 0.5 µl EcoRI enzyme, 1 µl 10X NEBuffer EcoRI, 5.5 µl nuclease-free water, and 3 µl purified plasmid DNA.
(d) Incubate digestion mixture at 37 °C for 1 h.
(e) During incubation, prepare a 1% agarose gel as described above in 4.4 steps (a–c).
(f) Add 2 µl 6X loading dye to each digest and load into agarose gel. Load 6 µl ladder into an empty well for sizing the ~3.5 kb vector and the 750-900 bp insert.
(g) Run agarose gel in 1X TAE buffer at 120 V for 15 min. Analyze the gel to calculate the frequency of clones with the desired insert (generally > 85%).

8. Bidirectional sequencing of cDNA clones

[DYEnamicTM ET Terminator Cycle Sequencing Kit (GE Healthcare), CleanSEQ_ Dye-Terminator Removal Kit (Agencourt), 85% ethanol (EtOH)]

(a) Prepare a 5 mM MgCl2, 20 mM Tris sequencing buffer by adding 0.102 g MgCl2 and 0.242 g Tris to 80 ml double-distilled water, stir to dissolve, adjust pH to 9.0, and bring volume to 100 ml with double-distilled water.
(b) Prepare sequencing reaction mix containing 1 µl DYEnamicTM ET Terminator, 1 µl 3.2 µM oligos, 6 µl sequencing buffer, 7 µl nuclease-free water, and 5 µl purified plasmid DNA. Two of the primers (M13R and T7) anneal directly to the pCR-Blunt vector. Two internal primers for each locus were designed using MHC II sequences of rhesus macaques (see table).
(c) Cycle sequence using the following thermocycler profile: 30 cycles of 95 °C for 20 s, 50 °C for 15 s, and 60 °C for 1 min.
(d) Purify the sequencing reactions using the Agencourt_ CleanSEQ_ Dye-Terminator Removal kit following the manufacturer’s protocol for ET Terminator clean-up.
(e) Resolve the cleaned sequencing products by capillary electrophoresis on an ABI 3730 Genetic Analyzer (Applied Biosystems), or a comparable Sanger sequencing platform.

Primers used to sequence MHC class II alleles:

9. Sequence analysis

Edit bidirectional sequencing results for high quality data using CodonCode Aligner (CodonCode Corporation) or similar sequence analysis software. Clip sequence ends to maximize the region with an error rate below 0.01 and delete short sequences with less than 50 Phred 20 quality bases. Approximately 15–30 clones were analyzed for the DPA, DPB, DQA, DQB, and DRA loci, while at least 48 clones were examined for the DRB loci. To avoid PCR artifacts, when three or more identical clones were found, the MHC class II sequence was considered to be an actual transcribed allele. Novel MHC class II sequences were deposited in GenBank.

10. Phylogenetic analyses

Sequences were aligned at the amino acid level using the CLUSTAL X program (Thompson et al. 1997), and the alignments were imposed on the DNA sequences. Phylogenetic trees were constructed by the neighbor-joining method (Saitou and Nei 1987) on the basis of the LogDet nucleotide distance. The reliability of clustering patterns in phylogenetic trees was assessed by bootstrapping (Felsenstein 1985); 1,000 bootstrap samples were used. To estimate divergence times of alleles, we used the linearized tree method (Takezaki et al. 1995) based on the number of synonymous nucleotide substitutions per synonymous site (Nei and Gojobori 1986). A calibration was provided by the estimate of Satta et al. (1993) of the rate of synonymous substitution at primate class II MHC loci.

Novel major histocompatability complex (MHC) allele discovery from cDNA amplicons by Roche/454 pyrosequencing – MHC II

1. Total RNA isolation from whole blood or cells

[MagNA Pure LC RNA Isolation Kit – High Performance (Roche Applied Science), alternatively other RNA isolation protocols may be utilized]

(a) Ensure that the MagNA Pure LC Instrument has the proper RNA purification protocols installed as detailed by the MagNA Pure LC RNA Isolation Kit manual.
(b) Prepare the isolation kit reagents as detailed in the manual.
(c) Aliquot 200 µl of whole blood or white blood cells, or 1 x 106 peripheral blood mononuclear or cultured cells as directed by the manual.
(d) Set up the instrument using the appropriate protocol for the starting material. Follow instructions supplied by the software, as it will calculate reagent volumes based upon number of samples to be isolated.
(e) Immediately upon completion of the isolation run, quantitate RNA samples with a NanoDropTM (Thermo Scientific) prior to proceeding with cDNA synthesis. If necessary, RNA samples can be stored in RNase-free microcentrifuge tubes at -80 °C.

2. Synthesis of first-strand cDNA

[MJ Research Tetrad Thermocycler (Bio-Rad Labortories) and Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen)], alternatively other thermocyclers may be utilized.

(a) Prepare a reaction mixture of 1 µl 10 mM dNTP mix, 1 µl 50 µM Oligo(dT)20, and 50 ng total RNA (or up to 8 µl if 50 ng RNA would exceed 8 µl). If necessary, add DEPC-treated water to obtain a 10 µl final reaction volume.
(b) Incubate reactions at 65 °C for 5 min to denature. During this incubation, prepare the cDNA synthesis master mix of 2 µl 10X RT Buffer, 4 µl 25 mM MgCl2, 2µl 0.1 M DTT, 1 µl RNaseOUTTM, and 1 µl SuperScriptTM III RT.
(c) Incubate initial reaction mixture at 4 °C or on ice for at least 1 min. During this incubation, add 10 µl cDNA synthesis master mix to initial reaction mixture.
(d) Incubate at 50 °C for 50 min.
(e) Terminate cDNA synthesis by incubating at 85 °C for 5 min.
(f) Add 1 µl 2U/µl E. coli RNase H to reaction mixture and incubate at 37 °C for 20 min to remove any remaining RNA.
(g) Upon completion, proceed directly to PCR or store cDNA at -20 °C.

3. PCR amplification of MHC class II cDNA

[PhusionTM High-Fidelity PCR Master Mix with HF Buffer (New England BioLabs), nuclease-free water, Flash- Gel DNA Cassette 1.2% (Lonza), FlashGel_ 5X loading dye (Lonza), FlashGel_ Quant Ladder 100–1.5 kb (Lonza)]

(a) Prepare 50 µl primer-specific reaction mixtures of 25 µl 2X PhusionTM High-Fidelity PCR Master Mix, 20 µl nuclease-free water, 1.25 µl 5 µM 5’ PCR primer, 1.25 µl 5 µM 3’ PCR primer, and 2.5 µl cDNA.
(b) PCR amplify using the following cycle conditions: 98 °C for 3 min, 29 cycles of 98 °C for 5 s, 63 °C for 1 s, 72 °C for 20 s, and a final elongation of 72 °C for 5 min.
(c) After 20 cycles, check a 4 µl aliquot of each reaction on a FlashGel DNA cassette (Lonza) following the manufacturer’s protocol. Add additional cycles to reactions as needed to obtain sufficient amplification of each sample.

Primers used to amplify MHC class II alleles:

4. Agarose gel electrophoresis and amplicon purification

[1X TAE, genetic analysis grade agarose, 6X loading dye, SYBR Safe DNA gel stain 10,000X concentrate in DMSO (Invitrogen), BenchTop 1 kb DNA ladder (Promega), MinElute Gel Extraction Kit (Qiagen)]


(a) Prepare a 1% agarose gel by mixing 150 ml 1X TAE with 1.5 g agarose.
(b) Heat the mixture in the microwave to melt the agarose, swirling occasionally.
(c) Add 15 µl SYBR_ Safe DNA gel stain to the mixture, swirling to mix. Pour gel and allow to solidify.
(d) Add 10 µl 6X loading dye to each PCR reaction and load entire volume into a single well of the agarose gel. Space samples appropriately leaving at least one empty well between samples to avoid cross-contamination. Load 6 µl ladder into an empty well for sizing of the amplicon.
(e) Run agarose gel in 1X TAE buffer at 120 V for 1 h. Ensure that the artifactual products have sufficiently separated from the amplicon of interest to allow for clean excision. Extend running time if necessary.
(f) Once sufficient separation is observed, visualize with a ‘DNA safe’ blue light transilluminator (Invitrogen) and excise the amplicon band. Place the gel slice into a colorless 1.5 ml microcentrifuge tube.
(g) Proceed with gel slice purification following the appropriate MinElute_ protocol. Elute the DNA with 10 µl nuclease-free water. Typical post-elution DNA concentrations range from 2–15 ng/ul.

5. Purified DNA quantitation and pooling

[Quant-iTTM dsDNA HS Assay Kit (Invitrogen), QubitTM assay tubes (Invitrogen)]

(a) Prepare the Quant-iTTM working solution, dilute standards and samples, calibrate the fluorometer, read samples, and calculate concentrations of samples as directed by the manufacturer’s protocol.
(b) If pooling products for ligation, normalize all products for each pool to the concentration of the least concentrated sample. Combine equal volumes of the normalized products to create each pool.
(c) Confirm purity of the pools on a High Sensitivity DNA Chip (Agilent) with a 2100 BioAnalyzer according to the manufacturer’s protocol.

6. Emulsion PCR

[EmPCR Reagents (Lib-L/A) (Roche), emPCR Oil and Breaking Kit (Roche), Ultra Turrax Tube Drive (IKA)]

6.1 Micro-emulsification

a) Vortex vigorously the 4 ml of emulsion oil and pour into the Turrax stirring tube
b) Prepare 1x Mock Mix. Add 430 μl of Mock Mix to 1.72 ml of Molecular Biology Grade (MBG) H2O
c) Add 2.0 ml of 1x Mock Mix to the stirring tube containing the mixed oil
d) Set the dial @ 4000 rpm for 5 minutes on the Turrax to make the microemulsions

6.2 Prepare Live Amplification Mix

a) Prepare the Live Amp Mix A (for Rapid and cDNA Rapid libraries) and B (for Paired End libraries). Add the reagents in the order they are listed in the table. The tube of Additive may show a precipitate. If this is observed, vortex well and heat the tube at 55°C for up to 5 minutes. If the precipitate remains, centrifuge the tube and use the supernatant.
b) Vortex the Live Amp Mix for 5 seconds, and store on ice.

A

B                       

6.3 Prepare Capture Bead

a) Take a frozen aliquot of Rapid Library double stranded library, thaw and vortex before denaturing it. Snap cool on ice.
b) Prepare 1x Wash Buffer. Add 0.5 ml of Wash Buffer to 4.5 ml of MGB H2O
c) Wash capture beads twice with 1.0 ml of 1x Wash Buffer
d) To calculate amount of library to add to beads:
(copies per bead desired * 10,000,000) / (molecules per μl of library)
Example: (1.5 cpb * 10E06 beads) / (2E06 mol/μl) = 7.5 μl library
Note: Prepare a dilution of the library such that the volume to be added will be between 1-20 μl
e) Remove supernatant completely and add denatured library at appropriate copies per bead and vortex

6.4 Macro-emulsification

a) Add 1.2 ml of Live Amp Mix to the capture bead, vortex, and transfer to the stirring tube (6 ml)
b) Set the dial @ 2000 rpm for 5 minutes on Turrax to make the macro-emulsions
c) Aliquot 100 μl into eight 8-strip cap tubes or a 96 well plate (total 64 wells)
Note: With the Combitip, slowly aspirate the emulsion taking care not to draw up air.

6.5 Thermocycling (~5 hours)

a) 94°C     4:00 min
b) 94°C     0:30 min
     58°C     4:30 min
     68°C     0:30 min
c) Go to step b 49 more times
d) 10°C forever

7. Breaking and Enrichment

[EmPCR Bead Recovery Reagents (Roche), Sequencing Kit Reagents and Enzymes (Roche)]

7.1 Dilute Reagents

a) Make 1x Melt solution (make fresh after 1 week). Add 125 μl of 10N NaOH to 10 ml MBG H20

7.2 Vacuum-assisted Emulsion Breaking Set Up

a) Fill the NUNC Immunowasher reservoir with isopropanol
b) Use a 8-channel NUNC Immuno Washer for dispensing isopropanol (or multichannel pipette)
c) Attach a 50 ml conical tube to the cap provided
d) Attach the blue connector to the transpette
e) Attach the other tubing to a waste trap connected to a vacuum.

7.3 Emulsion Breaking, Bead Washing and Recovery

a) Turn vacuum on
b) Place transpette into emulsions slowly in a circular manner to aspirate the emulsions, flip transpette upside down to promote aspiration
c) Add 100 μl isopropanol to wells, do not overfill the wells with isopropanol
d) Repeat steps 3b-3c for a total of two washes
e) After the 8-strip cap tubes have been processed, SLOWLY aspirate 5 ml isopropanol from a multi-channel reservoir to clean beads from tubing (quick aspiration leads to splashing and loss of beads!)
f) Turn vacuum off
g) Bring final volume to 35 ml with isopropanol in the 50 ml conical tube. Vortex to resuspend the beads before centrifugation
h) Spin in a table top centrifuge (i.e. Allegra 6 centrifuge) at 2000 rpm (930 *g) for 5 minutes (Make sure you balance the centrifuge)
i) Decant the supernatant without disturbing the white pellet. Add 10 ml of Enhancing Buffer to the 50 ml conical tube and vortex to resuspend the pellet. Bring the final volume to 40 ml with isopropanol and vortex. If necessary use a glass stirring rod or pipette to break up the pellet as much as possible
j) Spin in a table top centrifuge (i.e. Allegra 6 centrifuge) at 2000 rpm (930 *g) for 5 minutes for a second wash
k) Decant the supernatant without disturbing the white pellet and add 35 ml of isopropanol in the 50 ml conical tube and vortex
l) Spin in a table top centrifuge (i.e. Allegra 6 centrifuge) at 2000 rpm (930 *g) for 5 minutes for a third wash
m) Decant the supernatant without disturbing the white pellet and add 35 ml of Ethanol in the 50 ml conical tube and vortex
n) Spin in a table top centrifuge (i.e. Allegra 6 centrifuge) at 2000 rpm (930 *g) for 5 minutes for a fourth wash
o) Decant the supernatant without disturbing the white pellet and add 35 ml of Enhancing Buffer
p) Vortex the tube and centrifuge (i.e. Allegra 6 centrifuge) at 2000 rpm (930 *g) for 5 minutes
q) Decant the supernatant SLOWLY leaving ~2 mls of Enhancing Buffer on beads (stop pouring when the white beads are disturbed)
r) Transfer the DNA beads to a 1.7 ml micro-centrifuge tube. Spin the microcentrifuge tube and discard supernatant to create space for additional transfers.
s) Rinse the 50 ml conical with 1 ml of Enhancing Buffer and transfer into the 1.7
ml micro-centrifuge tube in step 3r.
t) Spin in centrifuge for 10 sec, stop, rotate tube 180° and centrifuge again for 10 sec to pellet DNA beads
u) Discard the supernatant. Add 1 ml of Enhancing Buffer and vortex.
v) Repeat steps 3t-3u for a total of two washes

7.4 Preparation for Indirect Enrichment

a) Discard the supernatant. Add 1 ml of 1x Melt solution to the 1.7 ml microcentrifuge tube and vortex. Incubate for 2 minutes at room temperature. Spin-rotate- spin and discard the supernatant.
b) Repeat step 4a for a total of two melts.
c) Discard the supernatant and add 1 ml of Annealing Buffer, vortex, spin
d) Discard supernatant and repeat step 4c twice for a total of three washes
e) Count the beads on the coulter counter for post-enrichment bead count
f) Remove the supernatant and add 45 μl of Annealing Buffer
g) Add 25 μl of Enrich primer to the tube, vortex to mix
h) Place tubes on a 65°C heat block for 5 minutes and cool on ice for 2 minutes
i) Spin, remove supernatant and wash with 1 ml Enhancing Buffer
j) Repeat step 4i twice for a total of three washes
k) Resuspend with 1 ml Enhancing Buffer, vortex, and set the beads aside for later

7.5 Preparation of Enrichment Beads

a) Vortex Enrichment beads (80 μl of enrichment beads) to resuspend completely
b) Place enrichment beads on MPC to pellet beads, wait 3 minutes and remove supernatant (the supernatant should be clear)
c) Add 1 ml of Enhancing Buffer to the tube, vortex, and pellet beads with MPC to remove the supernatant
d) Repeat step 5c for a total of two washes
e) Remove tube from the MPC and add 80 μl Enhancing Buffer and vortex

7.6 Enrichment of the DNA Carrying Beads

a) Pipette 80 μl of washed Enrichment beads into the 1.7 ml micro-centrifuge tube from step 4k above, vortex to mix completely
b) Rotate on a LabQuake tube roller at ambient temperature for 5 minutes
c) Place the tube on the MPC for 3-5 minutes for the enrichment beads to form a pellet on the magnet. The solution will appear white at this time. Invert the MPC a few times prior removal of the supernatant
d) Wash with 1 ml of Enhancing Buffer, VORTEX BETWEEN WASHES until beads are not observed in the supernatant (~6-10 washes). (Note: can collect supernatant into new 1.7 ml micro-centrifuge tube and briefly spin to determine if beads are still in supernatant)

7.7 Collection of the Enriched sstDNA Beads

a) Remove tube from the MPC and resuspend the bead pellet in 700 μl of 1x Melt solution, vortex, and place back on the MPC (note: do not leave beads in melt for longer than 10 minutes)
b) Collect the SUPERNATANT from the tube containing enriched sstDNA beads to a new 1.7 ml micro-centrifuge tube, spin collection tube and remove supernatant
c) Repeat step 7a and pool the two melts (total 1.4 ml) in the same collection tube
d) Discard the tube of spent Enrichment beads
e) Pellet the enriched sstDNA beads from step 7c by centrifugation
f) Discard the supernatant, and wash the enriched sstDNA beads three times with 1ml of Annealing Buffer to completely neutralize the melt solution
g) Remove and discard the supernatant without disturbing the pellet
h) Add 100 μl Annealing Buffer to the tube

7.8 Sequencing Primer Annealing

a) Add 25 μl of Seq Primer to the tube, vortex
b) Place tube on 65°C heat block for 5 minutes and cool on ice for 2 minutes
c) Spin, remove supernatant and wash with 1 ml of Annealing Buffer
d) Repeat step 8c twice for a total of three washes
e) Resuspend in 1 ml of Annealing Buffer
f) Count the beads for final enrichment bead count (Appendix 6). Repeat emulsions if you enrich above 2 million beads
g) The beads are ready for sequencing

8. Bead deposition

[Picotiter Plate (Roche), Bead Deposition Device (Roche), GS Junior (Roche)]

8.1 Common Buffer and Bead Buffer Preparation

a) Add 6.6 ml Supplement CB to Common Buffer (CB) bottle and mix bottle thoroughly. This is now Bead Buffer 1 (BB1)
b) Transfer 40 ml of BB1 to a 50ml falcon tube, add 6.5 µl of Apyrase, mix bottle thoroughly by inverting 10 times and leave buffer on ice. This is now Bead Buffer 2 (BB2)
c) Add 1 ml of DTT and 44 ml of Substrate Reagent to BB1. Mix bottle thoroughl by inverting 10 times.

8.2 Picotiter Plate (PTP) Preparation

a) Assemble a clean Bead Deposition Device (BDD) with a gasket and PTP
b) Load 350 µl of BB2 and spin PTP for 5min at 1640 rcf

8.3 Incubate DNA beads in BB2

a) In a 1.5 ml tube: add 500,000 sample DNA beads to 6 µl of Control Beads

8.4 Bead preparation

a) Packing Bead Preparation: Wash beads 3 times in 1 ml BB2 and resuspend in 200 µl of BB2. Centrifuge each wash for 5 min at 10,000 rpm
b) Enzyme/PPiase Bead Preparation: Wash enzyme beads and PPiase beads 3 times in 1 ml of BB2 using a magnetic particle collector. Resuspend enzyme beads in 400 µl of BB2 and resuspend PPiase beads in 410 µl of BB2. Prepare two labeled 1.7 ml tubes for two enzyme bead layers as follows and leave those tubes on ice:

Regions    BB2    Enzyme Beads    Total
Enzyme Prelayer    300    110    410
Enzyme Postlayer    180    230    410

c) Prepare DNA bead incubation: Spin DNA beads in table-top centrifuge for 10 seconds to pellet beads. Remove appropriate amount of BB2 from DNA sample to leave 50 µl volume. Add the following bead incubation reagents directly to DNA beads: 40 µl Polymerase, 20 µl Cofactor and 65 µl BB2. Vortex gently and incubate 10 min at room temperature on rotator.
d) After DNA incubation, add 175 µl of resuspended packing beads directly to the DNA mixture. Incubate the bead mixture on rotator for 5 min before loading

8.5 Bead Deposition: Enzyme Bead Prelayer (Layer 1)

a) Remove BB2 from soaking PTP
b) Vortex Enzyme Bead Prelayer and load 350 µl onto PTP
c) Spin PTP for 5 min at 1640 rcf

8.6 Bead Deposition: DNA + Packing Beads (Layer 2)

a) After Enzyme Bead Prelayer spin, carefully remove and discard the supernatant
b) Vortex DNA + Packing beads to mix thoroughly
c) Load 350 µl of DNA bead mixture onto PTP
d) Spin PTP for 10 min at 1640 rcf

8.7 Bead Deposition: Enzyme Bead Postlayer (Layer 3)

a) Carefully remove the supernatant from the DNA + Packing bead layer
b) Vortex Enzyme Bead Postlayer and load 350 µl onto the PTP
c) Spin PTP for 10 min at 1640 rcf

8.8 Bead Deposition: PPiase Bead Layer (Layer 4)

a) Remove and discard enzyme bead Postlayer supernatant
b) Vortex PPiase beads and load 350 µl onto PTP
c) Spin PTP for 5 min at 1640 rcf (if the instrument is not ready to load the PTP, spin the PTP for an extra 5 minutes. Repeat spin as needed.

8.9 Load PTP

a) After PPiase layer spin, remove and discard the supernatant
b) Disassemble BDD and immediately load PTP onto rig
c) After securing PTP in camera cartridge, gently wipe the smooth side of the PTP once with a dry kimwipe to remove excess liquid
d) Close the camera cartridge
e) Start sequencing script

Novel major histocompatability complex (MHC) allele discovery from cDNA amplicons by Roche/454 pyrosequencing – MHC I

1. Total RNA isolation from whole blood or cells

[MagNA Pure LC RNA Isolation Kit – High Performance (Roche Applied Science), alternatively other RNA isolation protocols may be utilized]

(a) Ensure that the MagNA Pure LC Instrument has the proper RNA purification protocols installed as detailed by the MagNA Pure LC RNA Isolation Kit manual.
(b) Prepare the isolation kit reagents as detailed in the manual.
(c) Aliquot 200 µl of whole blood or white blood cells, or 1 x 106 peripheral blood mononuclear or cultured cells as directed by the manual.
(d) Set up the instrument using the appropriate protocol for the starting material. Follow instructions supplied by the software, as it will calculate reagent volumes based upon number of samples to be isolated.
(e) Immediately upon completion of the isolation run, quantitate RNA samples with a NanoDropTM (Thermo Scientific) prior to proceeding with cDNA synthesis. If necessary, RNA samples can be stored in RNase-free microcentrifuge tubes at -80 °C.

2. Synthesis of first-strand cDNA

[MJ Research Tetrad Thermocycler (Bio-Rad Labortories) and Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen)], alternatively other thermocyclers may be utilized.

(a) Prepare a reaction mixture of 1 µl 10 mM dNTP mix, 1 µl 50 µM Oligo(dT)20, and 50 ng total RNA (or up to 8 µl if 50 ng RNA would exceed 8 µl). If necessary, add DEPC-treated water to obtain a 10 µl final reaction volume.
(b) Incubate reactions at 65 °C for 5 min to denature. During this incubation, prepare the cDNA synthesis master mix of 2 µl 10X RT Buffer, 4 µl 25 mM MgCl2, 2µl 0.1 M DTT, 1 µl RNaseOUTTM, and 1 µl SuperScriptTM III RT.
(c) Incubate initial reaction mixture at 4 °C or on ice for at least 1 min. During this incubation, add 10 µl cDNA synthesis master mix to initial reaction mixture.
(d) Incubate at 50 °C for 50 min.
(e) Terminate cDNA synthesis by incubating at 85 °C for 5 min.
(f) Add 1 µl 2U/µl E. coli RNase H to reaction mixture and incubate at 37 °C for 20 min to remove any remaining RNA.
(g) Upon completion, proceed directly to PCR or store cDNA at -20 °C.

3. Generation of primary cDNA-PCR amplicons

[PhusionTM High-Fidelity PCR Master Mix with HF Buffer (New England BioLabs), nuclease-free water, Flash- Gel DNA Cassette 1.2% (Lonza), FlashGel_ 5X loading dye (Lonza), FlashGel_ Quant Ladder 100–1.5 kb (Lonza)]

(a) Prepare 50 µl primer-specific reaction mixtures of 25 µl 2X PhusionTM High-Fidelity PCR Master Mix, 20 µl nuclease-free water, 1.25 µl 5 µM 5’ PCR primer, 1.25 µl 5 µM 3’ PCR primer (see below), and 2.5 µl cDNA.
(b) PCR amplify using the following cycle conditions: 98 °C for 3 min, 23 cycles of 98 °C for 5 s, 57 °C for 1 s, 72 °C for 20 s, and a final elongation of 72 °C for 5 min. For (c) After 23 cycles, check a 4 µl aliquot of each reaction on a FlashGel DNA cassette (Lonza) following the manufacturer’s protocol. Add additional cycles to reactions as needed to obtain sufficient amplification of each sample.

Sense-strand PCR primers

GS-FLX-A adaptor MIDx Tag Class I-specific:

Antisense-strand PCR primers

GS-FLX-B adaptor MIDx Tag Class I-specific:

4. Amplicon purification

[AMPure XP purification beads (Beckman Coulter), DynaMag-96 side skirted magnet (Invitrogen) or similar magnet plate, 1X TAE]

(a) Transfer 45 μl of sample to a 300 μl round bottom plate.
(b) Gently shake the AMPure XP bottle to resuspend any magnetic particles that may have settled.
(c) Add 45 μl AMPure XP beads (1:1 reaction volume to bead volume). Pipette 10 times to mix.
(d) Incubate for 5 min at room temp.
(e) Place reaction tube onto SPRI Plate Super Magnet Plate for 2 min to pellet beads.
(f) Aspirate clear supernatant from the reaction plate while still on magnet and discard.
(g) Dispense 200 μl of 70% ethanol to reaction plate while still on magnet, incubate for 30 sec at room temp, and aspirate out ethanol and discard.
(h) Repeat step (g) for a second ethanol wash.
(i) Remove reaction plate from the magnet. Add 30 μl of 1x TAE and pipette up & down 10 times to resuspend the beads.
(j) Incubate for 2 min at room temp.
(k) Place the reaction plate back on the magnet for 1 min to pellet the beads, and transfer supernatant to a new plate (make sure no beads are transferred).
(l) Repeat steps (b) through (k), adding 30 μl AMPure XP beads to PCR reaction (maintaining a 1:1 reaction volume to bead volume), for a second AMPure XP clean-up (can repeat more times if desired).

5. Purified DNA quantitation and pooling

[Quant-iTTM dsDNA HS Assay Kit (Invitrogen), QubitTM assay tubes (Invitrogen), Agilent High Sensitivity DNA Kit (Agilent Technologies), Bioanalyzer 2100 (Agilent Technologies)]

(a) Prepare the Quant-iTTM working solution, dilute standards and samples, calibrate the fluorometer, read samples, and calculate concentrations of samples as directed by the manufacturer’s protocol.
(b) If pooling products for ligation, normalize all products for each pool to the concentration of the least concentrated sample. Combine equal volumes of the normalized products to create each pool.
(c) Confirm purity of the pools on a High Sensitivity DNA Chip (Agilent) with a 2100 BioAnalyzer according to the manufacturer’s protocol.

6. Emulsion PCR

[EmPCR Reagents (Lib-L/A) (Roche), emPCR Oil and Breaking Kit (Roche), Ultra Turrax Tube Drive (IKA)]

6.1 Micro-emulsification

a) Vortex vigorously the 4 ml of emulsion oil and pour into the Turrax stirring tube
b) Prepare 1x Mock Mix. Add 430 μl of Mock Mix to 1.72 ml of Molecular Biology Grade (MBG) H2O
c) Add 2.0 ml of 1x Mock Mix to the stirring tube containing the mixed oil
d) Set the dial @ 4000 rpm for 5 minutes on the Turrax to make the microemulsions

6.2 Prepare Live Amplification Mix

a) Prepare the Live Amp Mix A (for Rapid and cDNA Rapid libraries) and B (for Paired End libraries). Add the reagents in the order they are listed in the table. The tube of Additive may show a precipitate. If this is observed, vortex well and heat the tube at 55°C for up to 5 minutes. If the precipitate remains, centrifuge the tube and use the supernatant.
b) Vortex the Live Amp Mix for 5 seconds, and store on ice.

A

B

 6.3 Prepare Capture Bead

a) Take a frozen aliquot of Rapid Library double stranded library, thaw and vortex before denaturing it. Snap cool on ice.
b) Prepare 1x Wash Buffer. Add 0.5 ml of Wash Buffer to 4.5 ml of MGB H2O
c) Wash capture beads twice with 1.0 ml of 1x Wash Buffer
d) To calculate amount of library to add to beads:
(copies per bead desired * 10,000,000) / (molecules per μl of library)
Example: (1.5 cpb * 10E06 beads) / (2E06 mol/μl) = 7.5 μl library
Note: Prepare a dilution of the library such that the volume to be added will be between 1-20 μl
e) Remove supernatant completely and add denatured library at appropriate copies per bead and vortex

6.4 Macro-emulsification

a) Add 1.2 ml of Live Amp Mix to the capture bead, vortex, and transfer to the stirring tube (6 ml)
b) Set the dial @ 2000 rpm for 5 minutes on Turrax to make the macro-emulsions
c) Aliquot 100 μl into eight 8-strip cap tubes or a 96 well plate (total 64 wells)
Note: With the Combitip, slowly aspirate the emulsion taking care not to draw up air.

6.5 Thermocycling (~5 hours)

a) 94°C     4:00 min
b) 94°C     0:30 min
     58°C     4:30 min
     68°C     0:30 min
c) Go to step b 49 more times
d) 10°C forever

7. Breaking and Enrichment

[EmPCR Bead Recovery Reagents (Roche), Sequencing Kit Reagents and Enzymes (Roche)]

7.1 Dilute Reagents

a) Make 1x Melt solution (make fresh after 1 week). Add 125 μl of 10N NaOH to 10 ml MBG H20

7.2 Vacuum-assisted Emulsion Breaking Set Up

a) Fill the NUNC Immunowasher reservoir with isopropanol
b) Use a 8-channel NUNC Immuno Washer for dispensing isopropanol (or multichannel pipette)
c) Attach a 50 ml conical tube to the cap provided
d) Attach the blue connector to the transpette
e) Attach the other tubing to a waste trap connected to a vacuum.

7.3 Emulsion Breaking, Bead Washing and Recovery

a) Turn vacuum on
b) Place transpette into emulsions slowly in a circular manner to aspirate the emulsions, flip transpette upside down to promote aspiration
c) Add 100 μl isopropanol to wells, do not overfill the wells with isopropanol
d) Repeat steps 3b-3c for a total of two washes
e) After the 8-strip cap tubes have been processed, SLOWLY aspirate 5 ml isopropanol from a multi-channel reservoir to clean beads from tubing (quick aspiration leads to splashing and loss of beads!)
f) Turn vacuum off
g) Bring final volume to 35 ml with isopropanol in the 50 ml conical tube. Vortex to resuspend the beads before centrifugation
h) Spin in a table top centrifuge (i.e. Allegra 6 centrifuge) at 2000 rpm (930 *g) for 5 minutes (Make sure you balance the centrifuge)
i) Decant the supernatant without disturbing the white pellet. Add 10 ml of Enhancing Buffer to the 50 ml conical tube and vortex to resuspend the pellet. Bring the final volume to 40 ml with isopropanol and vortex. If necessary use a glass stirring rod or pipette to break up the pellet as much as possible
j) Spin in a table top centrifuge (i.e. Allegra 6 centrifuge) at 2000 rpm (930 *g) for 5 minutes for a second wash
k) Decant the supernatant without disturbing the white pellet and add 35 ml of isopropanol in the 50 ml conical tube and vortex
l) Spin in a table top centrifuge (i.e. Allegra 6 centrifuge) at 2000 rpm (930 *g) for 5 minutes for a third wash
m) Decant the supernatant without disturbing the white pellet and add 35 ml of Ethanol in the 50 ml conical tube and vortex
n) Spin in a table top centrifuge (i.e. Allegra 6 centrifuge) at 2000 rpm (930 *g) for 5 minutes for a fourth wash
o) Decant the supernatant without disturbing the white pellet and add 35 ml of Enhancing Buffer
p) Vortex the tube and centrifuge (i.e. Allegra 6 centrifuge) at 2000 rpm (930 *g) for 5 minutes
q) Decant the supernatant SLOWLY leaving ~2 mls of Enhancing Buffer on beads (stop pouring when the white beads are disturbed)
r) Transfer the DNA beads to a 1.7 ml micro-centrifuge tube. Spin the microcentrifuge tube and discard supernatant to create space for additional transfers.
s) Rinse the 50 ml conical with 1 ml of Enhancing Buffer and transfer into the 1.7
ml micro-centrifuge tube in step 3r.
t) Spin in centrifuge for 10 sec, stop, rotate tube 180° and centrifuge again for 10 sec to pellet DNA beads
u) Discard the supernatant. Add 1 ml of Enhancing Buffer and vortex.
v) Repeat steps 3t-3u for a total of two washes

7.4 Preparation for Indirect Enrichment

a) Discard the supernatant. Add 1 ml of 1x Melt solution to the 1.7 ml microcentrifuge tube and vortex. Incubate for 2 minutes at room temperature. Spin-rotate- spin and discard the supernatant.
b) Repeat step 4a for a total of two melts.
c) Discard the supernatant and add 1 ml of Annealing Buffer, vortex, spin
d) Discard supernatant and repeat step 4c twice for a total of three washes
e) Count the beads on the coulter counter for post-enrichment bead count
f) Remove the supernatant and add 45 μl of Annealing Buffer
g) Add 25 μl of Enrich primer to the tube, vortex to mix
h) Place tubes on a 65°C heat block for 5 minutes and cool on ice for 2 minutes
i) Spin, remove supernatant and wash with 1 ml Enhancing Buffer
j) Repeat step 4i twice for a total of three washes
k) Resuspend with 1 ml Enhancing Buffer, vortex, and set the beads aside for later

7.5 Preparation of Enrichment Beads

a) Vortex Enrichment beads (80 μl of enrichment beads) to resuspend completely
b) Place enrichment beads on MPC to pellet beads, wait 3 minutes and remove supernatant (the supernatant should be clear)
c) Add 1 ml of Enhancing Buffer to the tube, vortex, and pellet beads with MPC to remove the supernatant
d) Repeat step 5c for a total of two washes
e) Remove tube from the MPC and add 80 μl Enhancing Buffer and vortex

7.6 Enrichment of the DNA Carrying Beads

a) Pipette 80 μl of washed Enrichment beads into the 1.7 ml micro-centrifuge tube from step 4k above, vortex to mix completely
b) Rotate on a LabQuake tube roller at ambient temperature for 5 minutes
c) Place the tube on the MPC for 3-5 minutes for the enrichment beads to form a pellet on the magnet. The solution will appear white at this time. Invert the MPC a few times prior removal of the supernatant
d) Wash with 1 ml of Enhancing Buffer, VORTEX BETWEEN WASHES until beads are not observed in the supernatant (~6-10 washes). (Note: can collect supernatant into new 1.7 ml micro-centrifuge tube and briefly spin to determine if beads are still in supernatant)

7.7 Collection of the Enriched sstDNA Beads

a) Remove tube from the MPC and resuspend the bead pellet in 700 μl of 1x Melt solution, vortex, and place back on the MPC (note: do not leave beads in melt for longer than 10 minutes)
b) Collect the SUPERNATANT from the tube containing enriched sstDNA beads to a new 1.7 ml micro-centrifuge tube, spin collection tube and remove supernatant
c) Repeat step 7a and pool the two melts (total 1.4 ml) in the same collection tube
d) Discard the tube of spent Enrichment beads
e) Pellet the enriched sstDNA beads from step 7c by centrifugation
f) Discard the supernatant, and wash the enriched sstDNA beads three times with 1ml of Annealing Buffer to completely neutralize the melt solution
g) Remove and discard the supernatant without disturbing the pellet
h) Add 100 μl Annealing Buffer to the tube

7.8 Sequencing Primer Annealing

a) Add 25 μl of Seq Primer to the tube, vortex
b) Place tube on 65°C heat block for 5 minutes and cool on ice for 2 minutes
c) Spin, remove supernatant and wash with 1 ml of Annealing Buffer
d) Repeat step 8c twice for a total of three washes
e) Resuspend in 1 ml of Annealing Buffer
f) Count the beads for final enrichment bead count (Appendix 6). Repeat emulsions if you enrich above 2 million beads
g) The beads are ready for sequencing

8. Bead deposition

[Picotiter Plate (Roche), Bead Deposition Device (Roche), GS Junior (Roche)]

8.1 Common Buffer and Bead Buffer Preparation

a) Add 6.6 ml Supplement CB to Common Buffer (CB) bottle and mix bottle thoroughly. This is now Bead Buffer 1 (BB1)
b) Transfer 40 ml of BB1 to a 50ml falcon tube, add 6.5 µl of Apyrase, mix bottle thoroughly by inverting 10 times and leave buffer on ice. This is now Bead Buffer 2 (BB2)
c) Add 1 ml of DTT and 44 ml of Substrate Reagent to BB1. Mix bottle thoroughl by inverting 10 times.

8.2 Picotiter Plate (PTP) Preparation

a) Assemble a clean Bead Deposition Device (BDD) with a gasket and PTP
b) Load 350 µl of BB2 and spin PTP for 5min at 1640 rcf

8.3 Incubate DNA beads in BB2

a) In a 1.5 ml tube: add 500,000 sample DNA beads to 6 µl of Control Beads

8.4 Bead preparation

a) Packing Bead Preparation: Wash beads 3 times in 1 ml BB2 and resuspend in 200 µl of BB2. Centrifuge each wash for 5 min at 10,000 rpm
b) Enzyme/PPiase Bead Preparation: Wash enzyme beads and PPiase beads 3 times in 1 ml of BB2 using a magnetic particle collector. Resuspend enzyme beads in 400 µl of BB2 and resuspend PPiase beads in 410 µl of BB2. Prepare two labeled 1.7 ml tubes for two enzyme bead layers as follows and leave those tubes on ice:

Regions    BB2    Enzyme Beads    Total
Enzyme Prelayer    300    110    410
Enzyme Postlayer    180    230    410

c) Prepare DNA bead incubation: Spin DNA beads in table-top centrifuge for 10 seconds to pellet beads. Remove appropriate amount of BB2 from DNA sample to leave 50 µl volume. Add the following bead incubation reagents directly to DNA beads: 40 µl Polymerase, 20 µl Cofactor and 65 µl BB2. Vortex gently and incubate 10 min at room temperature on rotator.
d) After DNA incubation, add 175 µl of resuspended packing beads directly to the DNA mixture. Incubate the bead mixture on rotator for 5 min before loading

8.5 Bead Deposition: Enzyme Bead Prelayer (Layer 1)

a) Remove BB2 from soaking PTP
b) Vortex Enzyme Bead Prelayer and load 350 µl onto PTP
c) Spin PTP for 5 min at 1640 rcf

8.6 Bead Deposition: DNA + Packing Beads (Layer 2)

a) After Enzyme Bead Prelayer spin, carefully remove and discard the supernatant
b) Vortex DNA + Packing beads to mix thoroughly
c) Load 350 µl of DNA bead mixture onto PTP
d) Spin PTP for 10 min at 1640 rcf

8.7 Bead Deposition: Enzyme Bead Postlayer (Layer 3)

a) Carefully remove the supernatant from the DNA + Packing bead layer
b) Vortex Enzyme Bead Postlayer and load 350 µl onto the PTP
c) Spin PTP for 10 min at 1640 rcf

8.8 Bead Deposition: PPiase Bead Layer (Layer 4)

a) Remove and discard enzyme bead Postlayer supernatant
b) Vortex PPiase beads and load 350 µl onto PTP
c) Spin PTP for 5 min at 1640 rcf (if the instrument is not ready to load the PTP, spin the PTP for an extra 5 minutes. Repeat spin as needed.

8.9 Load PTP

a) After PPiase layer spin, remove and discard the supernatant
b) Disassemble BDD and immediately load PTP onto rig
c) After securing PTP in camera cartridge, gently wipe the smooth side of the PTP once with a dry kimwipe to remove excess liquid
d) Close the camera cartridge
e) Start sequencing script

1. Genomic DNA isolation from whole blood or cells

To facilitate rapid genomic DNA isolation from fresh whole blood or frozen cell samples from up to 32 animals simultaneously, we use a MagNA Pure robot and MagNA Pure LC DNA isolation kit according to instructions provided by Roche Applied Science (https://www.roche-applied-science.com). Other DNA isolation techniques such as QIAamp DNA Blood Mini kits or DNeasy Blood and Tissue Kits from Qiagen (http://www.qiagen.com) can be substituted if this robot is not available. Regardless of isolation technique, proper protective equipment should be worn when handling nonhuman primate samples and DNA isolation reagents. Typical DNA concentrations from 300ul blood samples with the MagNA Pure LC DNA Isolation Kit range between 10–35 ng/ulin an elution volume of 100 ul.

(a) Ensure that the MagNA Pure LC Instrument has the proper DNA purification protocols installed as detailed by the MagNA Pure LC DNA Isolation Kit manual.

(b) Prepare the isolation kit reagents as detailed in the manual (https://www.roche-applied-science.com).

(c) Aliquot 300 ul of whole blood or white blood cells, or ~1 x 10⁶ peripheral blood mononuclear or cultured cells/100 ul as directed by the manual.

(d) Set up the instrument using the appropriate protocol for the starting material. Instructions supplied by the software will calculate individual reagent volumes based upon number of samples to be isolated.

(e) Upon completion of the isolation run, quantify DNA concentrations with a NanoDrop^TM (Thermo Scientific) normalize DNA samples to 15 ng/ul prior to proceeding with PCR-SSP amplification.  If necessary, samples can be stored indefinitely at -80 °C prior to PCR.

2. PCR-SSP amplification of Indian rhesus macaque MHC class I genomic DNA

[MJ Research Tetrad Thermocycler (Bio-Rad Laboratories), Thermal Activated Platinum Taq DNA Polymerase (100 μL, 5 units/μL) Invitrogen #10966034, Sterile Nuclease (DNase, RNase)-Free Water (Fisher Scientific #NC9628830)  alternatively other thermocyclers may be used]

(a) Aliquot 12 μL of appropriate primers into strip-cap PCR tubes (see Indian rhesus PCR-SSP primers under Primer Information tab).

(b) Aliquot total volume of PCR mix (components and recipes below) needed for the experiment into a 1.5 mL tube containing the PCR mix (8 μL PCR mix per PCR reaction).

(c) Add 0.6 uL  Platinum Taq for each 40 μL PCR mix to the 1.5 mL tube containing the PCR mix. DO NOT VORTEX but pipet gently up and down to mix.

(d) Add the Taq/PCR mix to the diluted DNA and mix each sample by pipetting up and down.

(e) Dispense 12 μL of DNA/PCR buffer into each of the wells containing primer, being careful to lay the drop on the side of the well near the top. Do not allow the pipet tip to come in contact with the well contents.

(f) Aliquot 8 μL of the remaining Taq/PCR mix in the 1.5 mL tube into each of the control wells.

(g) Aliquot 5 μL of the positive control Macaca mulatta gDNA at 15 ng/μL into the appropriate wells.

(h) Firmly seal the tray or tubes.

(i) Vortex sealed tray or strip tubes and pulse spin down.

(j) Place on thermocycler and run program.

Thermocycling conditions:

96°C initial denaturation for 1 min

6 cycles

96°C denaturation for 25 s

67.9°C annealing for 50 s           

72.0°C elongation for 45 s

6 cycles

96.0°C denaturation for 25 s

66.4°C annealing for 50 s          

72.0°C elongation for 45 s

5 cycles:

96°C denaturation for 25 s

66.0°C annealing for 60 s (during which annealing temp is decreased 1.0°C for each of the five cycles)

72.0°C elongation for 45 s

16 cycles:

96.0°C denaturation for 25 s

63.0°C annealing for 50 s

72°C elongation at 45 s

72°C final extension for 10 min

25°C terminal hold

2. Analysis by gel electrophoresis

When cycling is completed, reactions are analyzed by gel electrophoresis using SB gels and buffer.

(a) Make a 2% agarose gel with 0.1 μg/mL of ethidium bromide in 0.5X SB.

(b) Load 4 μL of DNA ladder into the first well of each lane of the gel.

(c) Load all 24 μL of the samples into wells.

(d) Run gel at 230 V for approximately 17 minutes.

(e) After running, take a picture with AlphaImager program (UV transillumination).

Protocol reference: Kaizu M, Borchardt, GJ, Glidden CE, Fisk DL, Loffredo JT, Watkins DI, Rehrauer WM. Molecular typing of major histocompatibility complex class I alleles in the Indian rhesus macaque which restrict SIV CD8+ T cell epitopes. Immunogenetics. 2007 Sep:59(9):693-703. Epub 2007 Jul 20.PMID: 17641866

Novel major histocompatability complex (MHC) allele discovery from cDNA libraries by Sanger sequencing – MHC class I

1. Bead preparation

[M-270 strepavidin Dyabeads (Invitrogen), Magna-Sep magnetic particle separator (Invitrogen)]

(a) Resuspend streptavidin bead stock by light vortexing

(b) Pipet 20 µl beads to 1.5ml microfuge tube for each capture

(c) Place in magnetic tube rack, wait for beads to move to side (~1 min), remove supernatant

(d) Wash beads by adding 100 µl RecA binding buffer

(e) Repeat steps 3-5 two more times for a total of 3 washes

(f) Resuspend beads in 30 µl RecA binding buffer

2. RecA capture

[RecA protein 2mg/ml (New England Biolabs), Adenosine 5’-triphosphate 100mM (Sigma-Aldrich), Adenosine 5’-[g-thiol] triphospate tetralithium salt (Sigma-Aldrich), Proteinase K (New England Biolabs), Phenylmethyl sulfonyl fluoride (PMSF) (Sigma-Aldrich)]

(a) Calculate the volume needed for 5µg cDNA from each library to be captured, total volume is 30µl

(b) Add the following reagents, in order, to a 1.5ml microcentrifuge tube for each capture

Probe: 5’-CGGAGATCAYRCTGACVTGGC-3’

The sequence of the capture oligonucleotide was derived from a highly conserved region of the MHC class I alpha-3 domain

(c) Mix well by pipetting, quick spin in a microcentrifuge

(d) Incubate for 15 min at 37°C

(e) Add appropriate volume of cDNA

(f) Incubate 20 min at 37°C

(g) Add 0.6 µl 10% SDS to each tube

(h) Add 0.5 µl Proteinase K to each tube

(i) Incubate 10 min at 37°C

(j) Add 1.0 µl PMSF (100mM, stored -20°C) to each tube

(k) Add 30 µl resuspended beads to each tube

(l) Incubate 30 min at room temperature, resuspending beads every 2 minutes by light tapping

(m) Place each tube in the magnetic separator for 2 minutes

(n) Remove supernatant

(o) Resuspend beads in 1 ml of RecA wash buffer, mix thoroughly by inverting for 1 minute

(p) Repeat steps (m)-(o)

(q) Repeat steps (m) and (n) for a total of 3 washes

(r) Add 1 ml of 37°C nuclease free H₂O and incubate for 5 minutes

(s) Repeat steps (m) and (o)

(t) Resuspend beads in 400 µl TE (10mM Tris,1mM EDTA, pH8.0)

3. PCI extraction

[Glycogen (20ug/ul) (Invitrogen), Phenol:Chloroform:Isoamyl alcohol 25:24:1 (Sigma-Aldrich)]

(a) Add 400 µl phenol:chloroform:isoamyl:alcohol (best done in a screw-top microcentrifuge tube)

(b) Vortex hard for 15 seconds

(c) Spin for 5 min at 14000g

(d) Remove the upper aqueous layer and transfer to fresh tube

(e) Magnetically separate any remaining beads and transfer to a new tube

(f) Add 100 µl 10M NH₄OAc; 1 µl glycogen, and 1ml of -20°C absolute ethanol

(g) Spin for 20 min at 14000g

(h) Carefully remove supernatant leaving pellet, wash with 500µl 70% EtOH, spin for 10 min at 14000g

(i) Remove supernatant and allow pellet to air dry

(j) Resuspend in 10 µl TE

4. Transformation

[DH5-a competent E.coli (Invitrogen)]

(a) Thaw 1 aliquot of DH5-a competent cells on ice

(b) Add 5 µl of RecA capture enriched cDNA from step 3 (j) and mix gently

(c) Incubate for 5 min on ice

(d) Heat shock for 30 sec at 42° C

(e) Incubate for 5 min on ice

(f) Add 250 µl of 37° SOC medium

(g) Incubate for 1 hr with shaking at 37°C

(h) Plate 75-150 µl per plate (15 cm petri dishes), grow overnight at 34° C.  

5. Plasmid isolation and purification

[Perfectprep Plasmid 96 Vac Direct Bind kit (Eppendorf),  CIRCLEGROW broth powder (Qbiogene)]

(a) Pick single colonies and grow overnight in 1ml CircleGrow broth with 100µg/ml ampicillin

(b) Isolate Plasmid cDNA according to the manufacturer’s protocol

6. Bidirectional sequencing of cDNA libraries

[DYEnamicTM ET Terminator Cycle Sequencing Kit (GE Healthcare), CleanSEQ_ Dye-Terminator Removal Kit (Agencourt), 85% ethanol (EtOH)]

(a) Prepare a 5 mM MgCl2, 20 mM Tris sequencing buffer by adding 0.102 g MgCl2 and 0.242 g Tris to 80 ml double-distilled water, stir to dissolve, adjust pH to 9.0, and bring volume to 100 ml with double-distilled water.

(b) Prepare sequencing reaction mix containing 1 µl DYEnamicTM ET Terminator, 1 µl 3.2 µM oligo (either 5’Refstrand or SBT190-R), 6 µl sequencing buffer, 7 µl nuclease-free water, and 5 µl purified plasmid DNA.

(c) Cycle sequence using the following thermocycler profile: 30 cycles of 95 °C for 20 s, 50 °C for 15 s, and 60 °C for 1 min.

(d) Purify the sequencing reactions using the Agencourt_ CleanSEQ_ Dye-Terminator Removal kit following the manufacturer’s protocol for ET Terminator clean-up.

(e) Resolve the cleaned sequencing products by capillary electrophoresis on an ABI 3730 Genetic Analyzer (Applied Biosystems), or a comparable Sanger sequencing platform.

Primers used to sequence MHC class II alleles:

Forward primers:

Reverse primers:

7. Sequence analysis

Sequences were analyzed using CodonCode Aligner version 1.6.3 (CodonCode, Deadham, MA). MHC class I alleles were considered part of the cDNA library after at least two copies were verified by sequencing. Novel MHC I sequences were given GenBank accession numbers.

8. Appendices

A. Probes:

Probes must be ≥40 nucleotides long.

They should be biotinylated at the 5' end and HPLC or PAGE purified

Resuspend at 100 ng/µl

B. 10X RecA capture buffer:

12.5 ml 1.0M Tris acetate pH 7.5

0.43 g MgOAc

0.5 g BSA

q.s. to 50 ml with H₂O

Final concentration for 10x: 250nM Tris Acetate, 40mM MgOAc, 10µg/µl BSA

C. ATP mixture:

2 parts 100 mM adenosine 5’-[g-thiol] triphosphate tetralithium salt (ATP gamma S)

1 part 100 mM adenosine triphosphate

D. RecA binding buffer:

2.92 g NaCl

500 µl 1M Tris Acetate, pH 7.5

100 µl 0.5 M EDTA, pH 8.0

50 ml millipore H₂O

Final Concentration: 10mM Tris, 1 mM EDTA, 1M NaCl

E. RecA wash buffer:

5.84 g NaCl

500 µl 1M Tris Acetate pH 7.5

100 µl 0.5 M EDTA pH 8.0

50 ml millipore H₂O

Final Concentration: 10mM Tris, 1 mM EDTA, 1M NaCl

Novel major histocompatability complex (MHC) allele discovery from cDNA libraries by Sanger sequencing – MHC class II

1. Bead preparation

[M-270 strepavidin Dyabeads (Invitrogen), Magna-Sep magnetic particle separator (Invitrogen)]

(a) Resuspend streptavidin bead stock by light vortexing

(b) Pipet 20 µl beads to 1.5ml microfuge tube for each capture

(c) Place in magnetic tube rack, wait for beads to move to side (~1 min), remove supernatant

(d) Wash beads by adding 100 µl RecA binding buffer

(e) Repeat steps 3-5 two more times for a total of 3 washes

(f) Resuspend beads in 30 µl RecA binding buffer

2. RecA capture

[RecA protein 2mg/ml (New England Biolabs), Adenosine 5’-triphosphate 100mM (Sigma-Aldrich), Adenosine 5’-[g-thiol] triphospate tetralithium salt (Sigma-Aldrich), Proteinase K (New England Biolabs), Phenylmethyl sulfonyl fluoride (PMSF) (Sigma-Aldrich)]

(a) Calculate the volume needed for 5µg cDNA from each library to be captured, total volume is 30µl

(b) Add the following reagents, in order, to a 1.5ml microcentrifuge tube for each capture

Probe used for MHC-II capture were the following:

DRA 5’-CAACGTCCTCATCTGTTTCATCGA-3’

DRB 5’-GCMCAGARCAAGATSCTGAGTGGW-3’

DQA 5’-SCGCARBKTGCACTGAGAAAC-3’

DQB 5’-TGTGCTACTWCRYCAACKGGA-3’

DPA 5’-TCTGGCATCTGGAGGAGTTTG-3’

DPB 5’-GCAGCTCTTTTCATTTTGCCATCC-3’

(c) Mix well by pipetting, quick spin in a microcentrifuge

(d) Incubate for 15 min at 37°C

(e) Add appropriate volume of cDNA

(f) Incubate 20 min at 37°C

(g) Add 0.6 µl 10% SDS to each tube

(h) Add 0.5 µl Proteinase K to each tube

(i) Incubate 10 min at 37°C

(j) Add 1.0 µl PMSF (100mM, stored -20°C) to each tube

(k) Add 30 µl resuspended beads to each tube

(l) Incubate 30 min at room temperature, resuspending beads every 2 minutes by light tapping

(m) Place each tube in the magnetic separator for 2 minutes

(n) Remove supernatant

(o) Resuspend beads in 1 ml of RecA wash buffer, mix thoroughly by inverting for 1 minute

(p) Repeat steps (m)-(o)

(q) Repeat steps (m) and (n) for a total of 3 washes

(r) Add 1 ml of 37°C nuclease free H₂O and incubate for 5 minutes

(s) Repeat steps (m) and (o)

(t) Resuspend beads in 400 µl TE (10mM Tris,1mM EDTA, pH8.0)

3. PCI extraction

[Glycogen (20ug/ul) (Invitrogen), Phenol:Chloroform:Isoamyl alcohol 25:24:1 (Sigma-Aldrich)]

(a) Add 400 µl phenol:chloroform:isoamyl:alcohol (best done in a screw-top microcentrifuge tube)

(b) Vortex hard for 15 seconds

(c) Spin for 5 min at 14000g

(d) Remove the upper aqueous layer and transfer to fresh tube

(e) Magnetically separate any remaining beads and transfer to a new tube

(f) Add 100 µl 10M NH₄OAc; 1 µl glycogen, and 1ml of -20°C absolute ethanol

(g) Spin for 20 min at 14000g

(h) Carefully remove supernatant leaving pellet, wash with 500µl 70% EtOH, spin for 10 min at 14000g

(i) Remove supernatant and allow pellet to air dry

(j) Resuspend in 10 µl TE

4. Transformation

[DH5-a competent E.coli (Invitrogen)]

(a) Thaw 1 aliquot of DH5-a competent cells on ice

(b) Add 5 µl of RecA capture enriched cDNA from step 3 (j) and mix gently

(c) Incubate for 5 min on ice

(d) Heat shock for 30 sec at 42° C

(e) Incubate for 5 min on ice

(f) Add 250 µl of 37° SOC medium

(g) Incubate for 1 hr with shaking at 37°C

(h) Plate 75-150 µl per plate (15 cm petri dishes), grow overnight at 34° C.  

5. Plasmid isolation and purification

[Perfectprep Plasmid 96 Vac Direct Bind kit (Eppendorf),  CIRCLEGROW broth powder (Qbiogene)]

(a) Pick single colonies and grow overnight in 1ml CircleGrow broth with 100µg/ml ampicillin

(b) Isolate Plasmid cDNA according to the manufacturer’s protocol

6. Single-pass cycle sequencing of cDNA clones

[SP6 primer (5’-GGCCTATTTAGGTGACACTATAG-3’, DYEnamicTM ET Terminator Cycle Sequencing Kit (GE Healthcare), CleanSEQ_ Dye-Terminator Removal Kit (Agencourt), 85% ethanol (EtOH)]

(a) Prepare a 5 mM MgCl2, 20 mM Tris sequencing buffer by adding 0.102 g MgCl2 and 0.242 g Tris to 80 ml double-distilled water, stir to dissolve, adjust pH to 9.0, and bring volume to 100 ml with double-distilled water.

(b) Prepare sequencing reaction mix containing 1 µl DYEnamicTM ET Terminator, 1 µl 3.2 µM oligo (either 5’Refstrand or SBT190-R), 6 µl sequencing buffer, 7 µl nuclease-free water, and 5 µl purified plasmid DNA.

(c) Cycle sequence using the following thermocycler profile: 30 cycles of 95 °C for 20 s, 50 °C for 15 s, and 60 °C for 1 min.

(d) Purify the sequencing reactions using the Agencourt_ CleanSEQ_ Dye-Terminator Removal kit following the manufacturer’s protocol for ET Terminator clean-up.

(e) Resolve the cleaned sequencing products by capillary electrophoresis on an ABI 3730 Genetic Analyzer (Applied Biosystems), or a comparable Sanger sequencing platform.

7. Sequence analysis

Sequences were analyzed using CodonCode Aligner version 1.6.3 (CodonCode, Deadham, MA). More than 100 macaque MHC-II clones were identified for each cDNA library, and then clones of interest were chosen for full-length sequencing.

8. Appendices

A. Probes:

Probes must be ≥40 nucleotides long.

They should be biotinylated at the 5' end and HPLC or PAGE purified

Resuspend at 100 ng/µl

B. 10X RecA capture buffer:

12.5 ml 1.0M Tris acetate pH 7.5

0.43 g MgOAc

0.5 g BSA

q.s. to 50 ml with H₂O

Final concentration for 10X: 250mM Tris Acetate, 40mM MgOAc, 10 µg/µl BSA

C. ATP mixture:

2 parts 100 mM adenosine 5’-[g-thiol] triphosphate tetralithium salt (ATP gamma S)

1 part 100 mM adenosine triphosphate

D. RecA binding buffer:

2.92 g NaCl

500 µl 1M Tris Acetate, pH 7.5

100 µl 0.5 M EDTA, pH 8.0

50 ml millipore H₂O

Final Concentration: 10mM Tris, 1 mM EDTA, 1M NaCl

E. RecA wash buffer:

5.84 g NaCl

500 µl 1M Tris Acetate pH 7.5

100 µl 0.5 M EDTA pH 8.0

50 ml millipore H₂O

Final Concentration: 10mM Tris, 1 mM EDTA, 1M NaCl

1. Total RNA isolation from whole blood or cells

[MagNA Pure LC RNA Isolation Kit – High Performance (Roche Applied Science), alternatively other RNA isolation protocols may be utilized]

(a) Ensure that the MagNA Pure LC Instrument has the proper RNA purification protocols installed as detailed by the MagNA Pure LC RNA Isolation Kit manual.

(b) Prepare the isolation kit reagents as detailed in the manual.

(c) Aliquot 200 µl of whole blood or white blood cells, or 1 x 106 peripheral blood mononuclear or cultured cells as directed by the manual.

(d) Set up the instrument using the appropriate protocol for the starting material. Follow instructions supplied by the software, as it will calculate reagent volumes based upon number of samples to be isolated.

(e) Immediately upon completion of the isolation run, quantify RNA samples with a NanoDropTM (Thermo Scientific) prior to proceeding with cDNA synthesis. If necessary, RNA samples can be stored in RNase-free microcentrifuge tubes at -80 °C.

2. Synthesis of first-strand cDNA

[MJ Research Tetrad Thermocycler (Bio-Rad Laboratories) and Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen), alternatively other thermocyclers may be utilized.]

(a) Prepare a reaction mixture of 1 µl 10 mM dNTP mix, 1 µl 50 µM Oligo(dT)20, and 50 ng total RNA (or up to 8 µl if 50 ng RNA would exceed 8 µl). If necessary, add DEPC-treated water to obtain a 10 µl final reaction volume.

(b) Incubate reactions at 65 °C for 5 min to denature. During this incubation, prepare the cDNA synthesis master mix of 2 µl 10X RT Buffer, 4 µl 25 mM MgCl2, 2µl 0.1 M DTT, 1 µl RNaseOUTTM, and 1 µl SuperScriptTM III RT.

(c) Incubate initial reaction mixture at 4 °C or on ice for at least 1 min. During this incubation, add 10 µl cDNA synthesis master mix to initial reaction mixture.

(d) Incubate at 50 °C for 50 min.

(e) Terminate cDNA synthesis by incubating at 85 °C for 5 min.

(f) Add 1 µl 2U/µl E. coli RNase H to reaction mixture and incubate at 37 °C for 20 min to remove any remaining RNA.

(g) Upon completion, proceed directly to PCR or store cDNA at -20 °C.

3. PCR-SSP amplification of Chinese rhesus macaque MHC class I cDNA

[AmpliTaq Gold PCR Master Mix (Applied Biosystems)]

(a) Prepare 20 μL primer-specific reaction mixtures of 10 μL AmpliTaq Gold PCR Master Mix, 2 μL 5 μM 5' PCR primer, 2 μL 5 μM 3' PCR primer, and 2 μL cDNA.

(b) PCR amplify using the following cycle conditions: denaturation at 96°C for 5 min; 35 cycles of 94°C for 30 s, optimal annealing temperature (65-69°C, see primer list for Chinese rhesus) for 45 s, 72°C for 45 s; and extension at 72°C for 10 min.

4. Agarose gel electrophoresis and amplicon purification

[1X TAE, genetic analysis grade agarose, 6X loading dye, SYBR Safe DNA gel stain 10,000X concentrate in DMSO (Invitrogen), BenchTop 1 kb DNA ladder (Promega), MinElute Gel Extraction Kit (Qiagen)]

(a) Prepare a 1% agarose gel by mixing 150 ml 1X TAE with 1.5 g agarose.

(b) Heat the mixture in the microwave to melt the agarose, swirling occasionally.

(c) Add 15 µl SYBR_ Safe DNA gel stain to the mixture, swirling to mix. Pour gel and allow to solidify.

(d) Add 4 µl 6X loading dye to each PCR reaction and load entire volume into a single well of the agarose gel. Space samples appropriately leaving at least one empty well between samples to avoid cross-contamination. Load 6 µl ladder into an empty well for sizing of     the amplicon.

(e) Run agarose gel in 1X TAE buffer at 120 V for 1 h. Ensure that the artifactual products have sufficiently separated from the amplicon to allow for clean excision. Extend running time if necessary.

(f) Once sufficient separation is observed, visualize with a ‘DNA safe’ blue light transilluminator (Invitrogen) and excise the amplicon band. Place the gel slice into a colorless 1.5 ml microcentrifuge tube.

(g) Proceed with gel slice purification following the appropriate MinElute protocol. Elute the DNA with 10 µl nuclease-free water. Typical post-elution DNA concentrations range from 2–15 ng/ul.

5. Sequence verification of positive products

[PCR-SSP oligos (0.8 μM), DYEnamicTM ET Terminator Cycle Sequencing Kit (GE Healthcare), CleanSEQ_ Dye-Terminator Removal Kit (Agencourt), 85% ethanol (EtOH)]

(a) Prepare a 5 mM MgCl₂, 20 mM Tris sequencing buffer by adding 0.102 g MgCl₂ and 0.242 g Tris to 80 mL double-distilled water, stir to dissolve, adjust pH to 9.0, and bring volume to 100 mL with double-distilled water.

(b) Prepare sequencing reaction mix containing 1 μL DYEnamic^TM ET Terminator, 1 μL 3.2 μM appropriate oligo, 6 μL sequencing buffer, 7 μL nuclease-free water, and 5 μL purified DNA.

(c) Cycle sequence using the following thermocycler profile: 30 cycles of 95°C for 20 s, 50°C for 15 s, and 60°C for 1 min.

(d) Purify the sequencing reactions using the Agencourt_CleanSEQ_Dye-Terminator Removal kit following the manufacturer's protocol for ET Terminator clean-up.

(e) Resolve the cleaned sequencing products by capillary electrophoresis on an ABI_3730 Genetic Analyzer (Applied Biosystems), or a comparable Sanger sequencing platform.

Reference:

Karl JA, et al. Identification of MHC class I sequences in Chinese-origin rhesus macaques. Immunogenetics. 2008 Jan;60(1):37-46. Epub 2007 Dec 21.

1. Total RNA isolation from whole blood or cells

[MagNA Pure LC RNA Isolation Kit – High Performance (Roche Applied Science), alternatively other RNA isolation protocols may be utilized]

(a) Ensure that the MagNA Pure LC Instrument has the proper RNA purification protocols installed as detailed by the MagNA Pure LC RNA Isolation Kit manual.

(b) Prepare the isolation kit reagents as detailed in the manual.

(c) Aliquot 200 µl of whole blood or white blood cells, or 1 x 106 peripheral blood mononuclear or cultured cells as directed by the manual.

(d) Set up the instrument using the appropriate protocol for the starting material. Follow instructions supplied by the software, as it will calculate reagent volumes based upon number of samples to be isolated.

(e) Immediately upon completion of the isolation run, quantify RNA samples with a NanoDrop^TM (Thermo Scientific) prior to proceeding with cDNA synthesis. If necessary, RNA samples can be stored in RNase-free microcentrifuge tubes at -80 °C.

2. Synthesis of first-strand cDNA

[MJ Research Tetrad Thermocycler (Bio-Rad Laboratories) and Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen), alternatively other thermocyclers may be utilized.]

(a) Prepare a reaction mixture of 1 µl 10 mM dNTP mix, 1 µl 50 µM Oligo(dT)20, and 50 ng total RNA (or up to 8 µl if 50 ng RNA would exceed 8 µl). If necessary, add DEPC-treated water to obtain a 10 µl final reaction volume.

(b) Incubate reactions at 65 °C for 5 min to denature. During this incubation, prepare the cDNA synthesis master mix of 2 µl 10X RT Buffer, 4 µl 25 mM MgCl2, 2µl 0.1 M DTT, 1 µl RNaseOUT^TM, and 1 µl SuperScriptTM III RT.

(c) Incubate initial reaction mixture at 4 °C or on ice for at least 1 min. During this incubation, add 10 µl cDNA synthesis master mix to initial reaction mixture.

(d) Incubate at 50 °C for 50 min.

(e) Terminate cDNA synthesis by incubating at 85 °C for 5 min.

(f) Add 1 µl 2U/µl E. coli RNase H to reaction mixture and incubate at 37 °C for 20 min to remove any remaining RNA.

(g) Upon completion, proceed directly to PCR or store cDNA at -20 °C.

3. PCR-SSP amplification of Mauritian cynomolgus macaque MHC class I cDNA

[Phusion Hot Start DNA polymerase (New England BioLabs)]

(a) Prepare 20 µl primer-specific reaction mixtures containing 1 µl cDNA, 0.5 µM F/R PCR-SSP primers, 0.4 U Phusion Hot Start DNA polymerase, high fidelity buffer, 2% DMSO (NEB) and 200 µM each dNTP (Roche).

(b) Use the the following cycle conditions: 98°C for 60 s; 25 cycles of 98C for 5 s, 67-69°C* for 1 s, 72°C for 20 s; 72°C for 5 minutes.

    *67°C for B*440101, B*460101, B*510101; 69°C for B*430101

4. Agarose gel electrophoresis and amplicon purification

[1x TAE, genetic analysis grade agarose, 6X loading dye, SYBR Safe DNA gel stain 10,000X concentrate in DMSO (Invitrogen), ø X174/HaeIII size ladder (Promega)]

(a) Prepare a 1.7% (w/v) agarose gel by combining 150 µl 1X TAE with 2.55 g agarose.

(b) Heat the mixture in the microwave to melt the agarose, swirling occasionally.

(c) Add 15 µl SYBR Safe DNA gel stain to the mixture, swirling to mix. Pour gel and allow to solidify.

(d) Add 4 µl 6X loading dye to each PCR reaction and load entire volume into a single well of the agarose gel. Space samples appropriately leaving at least one empty well between samples to avoid cross-contamination. Load 6 µl ladder into an empty well for sizing of     the amplicon.

(e) Run agarose gel in 1X TAE buffer at 120 V for 1 h. Ensure that the products have sufficiently separated. Extend running time if necessary.

(f) Once sufficient separation is observed, visualize with AlphaImager program (UV transillumination)

Reference:

Mee ET, et al. MHC haplotype frequencies in a UK breeding colony of Mauritian cynomolgus macaques mirror those found in a distinct population from the same geographic origin. J Med Primatol. 2009 Feb;38(1):1-14. Epub 2008 Nov 5.

Karl JA, et al. Identification of MHC class I sequences in Chinese-origin rhesus macaques. Immunogenetics. 2008 Jan;60(1):37-46. Epub 2007 Dec 21.