Direct Sequencing of Proliferating T Cell CDR3s

To directly sequence the CDR3s from proliferating T cells assayed by Seq-Well, we applied a recently published TCR pulldown method^{56 (link)} to WTA products from the 2-week, 3-week and 4-week timepoint samples from all four participants. Briefly, biotinylated capture probes from the TRBC region were annealed to melted WTA cDNA. Magnetic streptavidin beads were then used to pull down cDNA enriched for TRBC; this cDNA was subsequently amplified using KAPA HiFi Mastermix (Kapa Biosystems) and purified using 0.75× SeraPure beads to select for 0.8–1-kb sized DNA fragments. To select for sequences with full CDR3 regions, a pool of V-region primers was used to further amplify sequences of interest. Step-out PCR was used to add sequencing handles and the resulting libraries were sequenced on a NextSeq 550 using a 150-cycle NextSeq kit with 148 cycles for Read 1 (CDR3) and 20 cycles for Index 1 (BC + UMI). Sequences of the primers used are available in Tu et al.^{56 (link)}. CDR3 consensus sequences were aligned and determined as outlined previously. Across the entire dataset, we detected ~50% of TCR-β chain CDR3s.