title: GSM9444971: IL-15C induced Virtual memory T cells (n=3); Mus musculus; RNA-Seq
library_construction_protocol: Using anti-CD8 microbeads, CD8 T cells were isolated from splenocytes of wild-type and IL-15C–treated C57BL/6 mice by magnetic-activated cell sorting. Purified CD8 T cells were then sorted into naïve (CD44-CD49d-) and virtual memory (CD44hiCD49d-) subsets using a BD FACS Aria III cell sorter. Total RNA was extracted using the Isolate II RNA Mini Kit. Only RNA samples with RIN > 7.0 were used for downstream library construction. Libraries were prepared using the Illumina TruSeq Stranded mRNA Sample Prep Kit. Poly-A–containing mRNA molecules were purified using poly-T–attached magnetic beads and fragmented under elevated temperature. First-strand cDNA synthesis was performed using SuperScript II reverse transcriptase and random primers, followed by second-strand synthesis using DNA Polymerase I, RNase H, and dUTP. After end repair, A-tailing, and adapter ligation, products were purified and enriched by PCR to generate final cDNA libraries. Libraries were quantified using KAPA Library Quantification Kits and quality-checked using Agilent TapeStation D1000 ScreenTape. Indexed libraries were sequenced on an Illumina NovaSeqX platform to generate 2×100 bp paired-end reads.