RNA-seq of human CAR T cells: spleen of tumor-bearing NSG mouse withpembrolizumab
Total RNA (1 g per sample) was used to generate sequencing libraries using NEBNext Ultra RNA Library Prep Kit for Illumina following manufacturers protocol. Polyadenylated mRNA was enriched from total RNA using poly-T oligo-attached magnetic beads. Purified mRNA was fragmented using divalent cations under elevated temperature, and first-strand cDNA synthesis was performed with random hexamer primers and M-MuLV Reverse Transcriptase. Second-strand cDNA synthesis was carried out using DNA Polymerase I and RNase H. Resulting double-stranded cDNA underwent end repair, A-tailing, and adaptor ligation. Size selection was performed to enrich fragments approximately 150200 bp in length using AMPure XP beads. Following USER enzyme treatment to prepare ligated fragments for PCR, libraries were amplified and purified. Final library quality and size distribution were assessed using the Agilent Bioanalyzer 2100 system.