title: bulkTCR-seq of Homo sapiens; Pool A peptide-stimulated T cell line
design: To expand vaccine pool-specific T cells, PBMCs were thawed and rested overnight at 37C in 24 well plates at 1x107 cells per well in HS+IL-7 media, DMEM (Life Technologies) medium containing 10% human serum (Sigma-Aldrich), 1% Pen strep (Life Technologies), 1% HEPES (Life Technologies), 1% MEM nonessential amino acids (Life Technologies), 1% sodium pyruvate (Life Technologies), and 0.1% of 2-mercaptoethanol (Life Technologies), and 20 ng mL IL-7 (Prepotech). The next day PBMCs were harvested and replated in 24 well plates at 1x107 cells per well and stimulated with 10µg mL of either Pool A, Pool B, or DMSO in HS+IL-7 media. Three days after peptide stimulation, half of the media in each well was removed and replaced with equal volume of an IL-2 containing media, HS+IL-7+IL-2, RPMI (Life Technologies) medium containing 10% human serum (Sigma-Aldrich), 1% Pen strep (Life Technologies), 1% HEPES (Life Technologies), 1% MEM nonessential amino acids (Life Technologies), 1% sodium pyruvate (Life Technologies), and 0.1% of 2-mercaptoethanol (Life Technologies), and 20 ng mL IL-7 (Prepotech), and 20 U mL of IL-2 (PeproTech). Half media replacement was performed every 3 days. Cultured cells were harvested 10-14 days post-stimulation, and T cells were isolated via CD3 magnetic bead enrichment (Miltenyi Biotec) following manufacturers protocol. Isolated T cells were then stored in Buffer RLT Plus (Qiagen) at -80C until RNA extraction. RNA from T cells was isolated using the Allprep DNA RNA kit (Qiagen) and stored in Nuclease-free water (Qiagen). Alpha and beta TCR repertoire analysis in bulk RNA samples was performed using rhTCRseq protocol published previously59. Specifically, 10-20ng bulk RNA was used in each RT reaction. After the sequencing library was made, it was sequenced using NextSeq P1-300 cycle kit on the Illumina NextSeq 1000 sequencing system according to the manufacturer's protocol with 248-nt read 1, 48-nt read 2, 8-nt index 1, and 8-nt index 2. The sequencing data analysis was done based on established method59.