GSM6998209: CD8_wt_8; Mus musculus; RNA-Seq
For the isolation of TILs, tumor tissue was dissociated with collagenase D (2.5mg/ml) at 37 degrees Celsius for 20 min, followed by centrifugation through a discontinuous Percoll gradient (GE Healthcare). 1,000 living CD8+ T cells (CD45+ TCRß+ CD8) were FACS-sorted immediately into TCL buffer (QIAGEN) supplemented with 1% ß-mercaptoethanol (Sigma Aldrich). Full-length RNA-seq library preparation was performed following a modified SMART-Seq2 protocol. cDNA concentration was quantified (Quant-iT PicoGreen dsDNA Assay Kit, Thermo Fisher Scientific) and normalized to 0.25ng/ul and libraries were prepared using the Nextera XT DNA Library Preparation kit (Illumina). Using a Bioanalyzer (Agilent), the final libraries were verified to have a size of 500 bp. For sequencing, the barcoded libraries were pooled, normalized to 2 nM, and denatured using 0.1 N NaOH. Sequencing was performed following the manufacturer's protocols using the paired-end Illumina sequencing (38bp X 2) with 75 cycle Nextseq 500 high output V2 kit (Illumina).