GSM8277633: P34-CP7-G12_S84_L004; Homo sapiens; RNA-Seq
These antigen-specific T cells were FACS-sorted into individual wells of a 96-well PCR plate with lysis buffers. To assess the technical variability, we mixed the lysis buffers with the external RNA control – ERCC RNA Spike-In (a final estimate of ~5000 molecules per well, Thermo Fisher Scientific, Waltham, MA). We used the modified Smart-Seq2 protocol (Clontech Laboratories, Mountain View, CA) to generate the cDNA library. From the library, we took a small aliquot (1 μL) for the nested PCR to amplify and sequence 15 targeted RNAs (TBX21, RORC, GATA3, FOXP3, RUNX1, RUNX3, IFNG, IL2, IL17A, GZMB, PERF, IL4, IL5, IL13, TGFB) and the CDR3 regions of both TCRα and TCRβ chains. Based on the TCR sequences, the Smart-seq2 generated cDNAs of the clonally expanded activated T cells were manually selected only on the ESAT-6/CFP-10-reactive cells for the high-coverage in-depth single-cell full transcriptomic sequencing. Nextera XT library prep kit (96 index primers; Illumina, San Diego, CA) protocol