Single-cell TCR sequencing

scPCR protocol for MAIT clonal analysis (NEB reagents)

1. Materials 
   1. NEB one Step RT-PCR kit
   2. NEB One Taq Hot Start DNA polymerase
   3. dNTPs (10 mM each)
2. Reaction 1 PCR
   1. Prepare reaction 1 primer mixture (10X concentration) (see primer list)
      1. Final concentration of each V region primer will be 0.06 uM, 10X concentration is thus 0.6 uM
      2. Final concentration of each constant region primer (alpha and beta) will be 0.3 uM, 10X concentration is thus 3 uM
      3. Final concentration of each phenotyping primer (forward and reverse) is 0.1 uM, 10X concentration is thus 1 uM
      4. As a sample mixture, to make enough for 1000 reactions, add 6 ul each 100 uM V region primer (37 in all, 1 alpha and 36 beta, 222 ul total), 30 ul 100 uM C alpha, 30 ul 100 uM C beta, 10 ul each 100 uM phenotyping primer except R1B_GATA3. For R1B_GATA3 add 11.6 (40 in all, 401.6ul total)  and nuclease-free water up to 1000 ul (316.4 ul)
   2. Prepare R1 reaction mixture (10 ul per cell), given volumes are given for a 1X reaction, using components from the NEB One Step RT-PCR kit
      1. 2X reaction mixture, 5 ul
      2. RT enzyme, 0.4 ul
      3. Reaction 1 10X primer mix, 1 ul
      4. nuclease free or DEPC H2O, 3.6 ul
   3. Add 10 ul per well into 96 well plates, seal plate and keep on ice until sorting
   4. Sort single cells into wells, add new seal, gently vortex and spin down at 500xg for 1 min
   5. Freeze down plate at -80 deg C until ready to run PCR
   6. After thawing plate, spin down, add new seal if necessary (ie if adhesive properties damaged by freezing and ice) and run PCR program
      1. 48 deg C 40 min
      2. 94 deg C 1 min
      3. 94 deg C 15 sec
      4. 62 deg C 1 min
      5. 68 deg C 1 min
      6. go to step iii, repeat 25 cycles
      7. 68 deg C 5 min
      8. 4 deg C hold indefinitely
   7. Freeze down plate or keep at 4 deg C if reaction 2 will take place within a day or two
3. Reaction 2 PCR
   1. Prepare reaction 2 primer mixture for TCR
      1. Final concentration of each V region primer will be 0.06 uM, 10X concentration is thus 0.6 uM
      2. Final concentration of each constant region primer (alpha and beta) will be 0.3 uM, 10X concentration is thus 3 uM
      3. As a sample mixture, to make enough for 1000 reactions, add 9 ul each 100 uM V region primer (37 in all, 1 alpha and 36 beta, 333 ul total), 45 ul 100 uM C alpha, 45 ul 100 uM C beta, and nuclease-free water up to 1500 ul (1077 ul)
   2. Prepare reaction 2 primer mixture for phenotyping genes
      1. Final concentration of each primer is 0.1 uM, 10X concentration is thus 1 uM
      2. As a sample mixture, to make enough for 1000 reactions, add 15 ul each R2 phenotyping primer except for R2A_GATA3. For R2A_GATA3, add 16.9uL (40 in all, 601.9 ul total) and nuclease-free water up to 1500 (898.1uL)
   3. Set up TCR and phenotyping reaction 2, using the NEB One Taq Hot Start DNA polymerase
      1. 5X standard buffer, 3 ul
      2. dNTP (10 mM each), 0.3 ul
      3. 10X TCR or phenotyping primer mix, 1.5 ul
      4. One Taq Hot Start DNA pol, 0.075 ul
      5. Template from Reaction 1 (1 ul, do not add to master mix)
      6. H2O up to 15 ul (9.125 ul)
   4. Add 14 ul reaction 2 master mix to each well
   5. Add 1 ul from reaction 1 to corresponding wells in reaction 2 for TCR and phenotyping
      1. May freeze plates for later PCR
   6. Run PCR program for reaction 2
      1. 94 deg C 30 sec
      2. 94 deg C 15 sec
      3. 64 deg C 1 min
      4. 68 deg C 1 min
      5. Go to step ii, repeat 25 cycles if TCR, 35 cycles if phenotyping
      6. 68 deg C 5 min
      7. 4 deg C hold indefinitely
   7. Freeze down plate or keep at 4 deg C if reaction 3 will take place within a day or two
4. Reaction 3 PCR (barcoding)
   1. Prepare working concentration of 5’ barcoding primers (0.375 uM)
   2. Prepare working concentration of 3’ barcoding primers (0.375 uM), TCR alpha and TCR beta reaction 3 will be separate, 3’ barcoding phenotyping primers at 0.375 uM
   3. If alpha and beta barcoding reaction will be combined (gives slightly lower efficiency, but saves time and PCR reagents), 3’ barcoding alpha concentration is 1.5 uM and 3’ barcoding beta conc is 0.375 uM
   4. Prepare barcoding master mix
      1. 5x standard buffer, 3 ul
      2. dNTP (10 mM each), 0.3 ul
      3. One Taq Hot Start DNA pol, 0.075 ul
      4. PE1 primer (100  uM), 0.075 ul
      5. PE2 primer (100 uM), 0.075 ul
      6. H2O, 6.475 ul
      7. template, 1 ul (to be added last, not in master mix)
      8. 5’ barcoding primer, working conc, 2 ul (not added to master mix)
      9. 3’ barcoding primer, working conc, 2 ul (not added to master mix)
   5. Using multichannel pipet, add 10 ul master mix to each well in 96 well PCR plate
   6. Using multichannel pipet, add 2 ul 5’ barcoding primers
      1. Barcoding primers can be added to separate 96 well plate arranged with each plate in one column, and each row in the respective row), for final concentration of 0.05 uM. Only need to change tips between plates
   7. Using multichannel pipet, add 2 ul 3’ barcoding primers for final concentration of 0.05 uM (if combined, 0.2 uM alpha and 0.05 uM beta)
      1. Place 3’ barcoding primers in 96 well plate, each column being a separate primer, ie primer 1 will be in rows A-H, column 1, primer 2 in rows A-H, column 2 etc
      2. TCR alpha and TCR beta barcoding done in separate plates
   8. Add 1 ul template from reaction 2
      1. Template for TCR alpha and TCR beta reactions come from TCR reaction 2, template for phenotyping reaction comes from phenotyping reaction 2
   9. Run PCR program for barcoding
      1. 94 deg C 30 sec
      2. 94 deg C 15 sec
      3. 66 deg C 30 sec
      4. 68 deg C 1 min
      5. Go to step ii, repeat 36 cycles
      6. 68 deg C 5 min
      7. 4 deg C hold indefinitely
   10. Freeze down plate or keep at 4 deg C if preparation for sequencing will take place within a day or two
5. Submission to MiSeq
   1. From each well of reaction 3, remove 5 ul using multichannel pipet and combine in reservoir (no need to change tips, everything will be mixed together in the end anyways)
   2. From mixture, run 50 ul on a 1.2% agarose gel, cut out and purify the band about 375 bp.
   3. Submit sample to sequencing core for MiSeq
   4. Process samples using attached VDJ_analysis_pipeline Perl script

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