TCR_8_48
Single cells suspended in PBS with 0.04% BSA were loaded on a Chromium Single Cell Instrument (10x Genomics). RNA-seq and V(D)J libraries were prepared using Chromium Single Cell 5 Library, Gel Beads & Multiplex Kit (10x Genomics). To enrich the V(D)J library aliquot for TCR a/b, the cDNA was split into two 20 ng aliquots and amplified in two rounds using primers designed in-house.  Specifically, for first round amplification the primers used were MP147 (ACACTCTTTCCCTACACGACGC) for short R1, MP122 (GGTGCTGTCCTGAGACCGAG) for mouse TRAC, or MP123(CAATCTCTGCTTTTGATGGCTCAAAC) for mouse TRBC. For second round amplification, 20 ng aliquots from the first round were amplified using MP147, MP130 (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT TGGTACACAGCAGGTTCTGG) a nested R2 plus mouse TRAC, or MP131 (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT GAC CTT GGG TGG AGT CAC ATT TCT C ) a nested R2 plus mouse TRABC. V(D)J libraries were prepared from 25 ng each mTRAC and mTRBC amplified cDNA. Paired-end sequencing was performed on Illumina NextSeq500 for RNA-seq (Read 1: 26-bp for UMI and cell barcode; 8-bp i7 sample index; and Read 2: 55-bp transcript read) and V(D)J libraries (Read 1: 150-bp; 8-bp i7 sample index; and Read 2: 150-bp). Data pre-processing was performed using Cell Ranger v2.0 and the mouse mm10 genome/reference for gene expression alignment and the IMGT-mouse genome/reference for V(D)J alignment.