GSM7520270: late, antibody derived trancript (ADT) library, CD3 sorted intracranial CT2A; Mus musculus; OTHER
CD3+ tumor infiltrates were isolated using FACS sorting. Single-cell suspensions were loaded on the 10x Genomics Chromium controller, and mRNA converted to cDNA libraries. Single-cell libraries were sequenced on a Novaseq 6000 150PE S-Prime flowcell. Libraries were sequenced in single index mode with the following read lengths: 28x8x91. 10x 5' Chromium chemistry GEMs are generated by combining barcoded Single Cell 5' Gel Beads, Master Mix, Partitioning Oil and single cells on a Chromium microfluidic Chip. Immediately following GEM generation, the Gel Bead is dissolved and any co-partitioned cell is lysed. Oligonucleotides containing an Illumina R1 sequence (read 1 sequencing primer), a 16 nt 10x Barcode, a 10 nt unique molecular identifier (UMI), and 13 nt template switch oligo (TSO) are released and mixed with the cell lysate and a Master Mix containing reverse transcription (RT) reagents and poly(dT) RT primers. Incubation of the GEMs produces 10x Barcoded, full-length cDNA from polyadenylated mRNA. GEMs are then broken and pooled and the 10x Barcoded first-strand cDNA is purified from the post GEM-RT reaction mixture and amplified. Full-length V(D)J segments (10x Barcoded) are enriched from amplified cDNA via PCR amplification with universal primer for 5' adapter and successive nested primers specific to the TCR and/or BCR constant regions. Enrichment and library preparation of the TCR and BCR constant regions takes place in two separate workflows using the same amplified cDNA stock. Enzymatic fragmentation and size selection are used to generate variable length fragments that collectively span the V(D)J segments of the enriched TCR and/or BCR transcripts prior to library construction. P5, P7, a dual sample index, 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 priming sites used in Illumina sequencing. Sequence is generated using Illumina short read paired end sequencing.