GSM7688516
Tumor dissected from animals and mechanically dissociated. Incubated with Collagenase I (in PBS Mg+Cl+) in a shaking incubator. Lymphocytes separated using lymphocyte separation media (LSM) density gradient. Collected cells stained for Live Dead and CD8 then sorted using FACSAria for viable CD8+ cells. 3 mice per replicate pooled together. Libraries were contructued according to the manufactures protocol for 10X Genomics single cell 5'v2. Isolated cells resuspended in master mix and placed in chip with gel beads and partitioning oil to create gel bed in emulsion (GEM)s. GEMs utilized for reverse transcription (RT) with addition of 10X barcode, unique molecular identifier (UMI), and R1 primer. cDNA amplified before preparation of libraries for gene expression. cDNA fragmented and size selected using SPRI beads for gene expression (GEX) library construction. Duringh library construction Illumina R2 primer, paired end constructs with P5 and P7, and sample index were added.