GSM4998200: P2_gex; Homo sapiens; RNA-Seq
Single-cell sequencing libraries were prepared using Chromium 10x Genomics platform with a V(D)J Enrichment Kit and 5' Library and Gel Bead Kit (10x Genomics, Pleasanton, CA, USA, PN-1000005, PN-1000006) according to the manufacturer's instructions The single-cell suspension of CD4+CD25hiCD127lo T-Tregs, B-Tregs, and CD4+CD45RA+CCR7+CD25loCD127hi B-naïve Tconvs from paired tumor and blood sorted using FACS Aria II was diluted in nuclease-free water and then combined with Single-Cell 5' Gel Beads, a master mix, and Partitioning Oil on Chromium Chip A, found in the aforementioned kit. RNA transcripts were uniquely barcoded and reverse-transcribed within nanoliter-scale droplets containing barcoded beads. 10x barcoded full-length cDNA were then pooled and enriched via PCR. For V(D)J Enriched Library, the enriched cDNA pool was amplified using primers specific to TCR. For 5'Gene Expression (GEX) library, the cDNA pool underwent an end repair process, A-tailing, adaptor ligation, and sample index PCR. Both libraries for scRNA-seq and scTCR-seq were sequenced using the HiSeqX platform (Illumina).