title: bulkTCR-seq of Homo sapiens- peripheral blood
design: PBMCs were  thawed and rested overnight at 37C in 24 well plates at 1×107 cells per well in HS+IL-7 media, DMEM (Life Technologies) medium containing 10% human serum (Sigma-Aldrich), 1% Penstrep (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 T cells were isolated via CD3 magnetic bead enrichment (Miltenyi Biotec, 130-050-101) 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.