GSM8996452: human.PBMCs.CD8_HTGTS_TCR_seq_TcrJ_mix_rep1; Homo sapiens; OTHER
Cells were collected and lysed in cell lysis buffer using proteinase K. Genomic DNA was later precipitated and dissolved in TE before shearing. 3-20 μg DNA was fragmented via sonication on Qsonic biorupter and subjected to linear PCR amplification with a biotinylated primer as following PCR conditions: 95°C 2 min; 95°C 30s; 58°C 30 s; 72°C 90 s; 80 cycles; 72°C 2 min. Primers were removed by VAHTS DNA Clean Beads (Vazyme, N411). The resulting single-strand biotinylated PCR products were collected via Dynabeads MyONE C1 streptavidin beads (ThermoFisher, 65002) and ligated to bridge adapters overnight in the presence of 15% PRG8000. Ligation were conducted following 25°C 60 min; 22°C 120 min; 16°C 9-12 h. Subsequently, i5-indexed bait-specific inner primers and i7-index adapter primers were added to adapter-ligated products by nested PCR. Excess primers were removed by VAHTS DNA Clean Beads (Vazyme, N411), and the purified PCR products were further tagged with Illumina sequencing adapter sequences. Libraries were size-selected by resolving PCR products on a 2% agarose gel, isolating fragments between 300-700 bp for mouse or human samples, with recovery performed via gel extraction. The HTGTS-TCR-seq libraries were sequenced on NovaXplus (Illumina) using 150-bp paired-end reads.