title: GSM8368416: NS, scTCR-seq; Mus musculus; OTHER
library_construction_protocol: Tumor tissues were dissected into 1~2 mm pieces with scissors in PBS buffer and digested in RPMI 1640 Medium containing 1% FBS, 1% penicillin-streptomycin, 1 mg/ml Collagenase II (Sigma) and 100 μg/ml DNase I (Sigma) at 37 °C for 30 min. Subsequently, tumor tissue cells were filtered with a 70 µm cell strainer (BD Biosciences). After a 5 min centrifugation at 500 g, cells were resuspended with red blood cell lysis buffer (Beyotime) to deplete red blood cells. Thereafter, cells were washed 2~3 times and kept in RMPI1640 medium with 1% FBS and 2mM EDTA at 4°C for cell sorting.Libraries were constructed using the Single Cell %' Library Kit V3 (10× Genomics). Enzymatic fragmentation and size selection are used to generate variable length fragments that collectively span the V(D)J segments of the amplified TCR and/or BCR transcripts prior to library construction. P5, P7, i5 and i7 sample indexes, and an Illumina R2 sequence (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and Sample Index PCR. The final libraries contain the P5 and P7 primers used in Illumina amplification.