GSM8516563: TCRb, ND602, Cells 49-96; Homo sapiens; RNA-Seq
CD8+ T cells and B cells were negatively enriched from cryopreserved PBMCs and cocultured in the presence of peptide, IL2, IL4, IL21, and multimeric CD40L. After 2 weeks, cells were stained with cognate pMHC tetramers and tetramer+ CD8+ T cells were single cell sorted on a BD AriaFusion into lysis buffer. Lysates were snap frozen and stored at -80℃ until use. Reverse transcription and cDNA amplification was done in single cell lysate without RNA extraction Reverse transcription was performed on lysates with TRBC- and TRAC-specific primers. PCR1 was carried out with the same TRBC/TRAC primers and V-region primers. PCR2 for alpha and beta chains was carried out separately with dual-nested primers binding constant and V-regions with partial Illumina adapters. Index PCR was carried out with Illumina P5 and P7 dual-index primers. Index PCR products for each cell were then pooled and gel purified.