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

Titanium Chemistry

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

Ti-A adaptor MIDx Tag Class I-specific:

Antisense-strand PCR primers

Ti-B adaptor MIDx Tag Class I-specific:

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 190 bp 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 190 bp 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 190 bp 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), 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