GSM8671330: WT Ly6C- Sca-1- Virtual memory T cells; Mus musculus; RNA-Seq
Using anti-CD8 microbeads, CD8 T cells were isolated from the C57BL/6 spleens by magnetic sorting and sorted into naïve (CD44-CD49d-), TM (CD44hiCD49d+) and VM (CD44hiCD49d-) on the basis of Ly6C and Sca-1 expression. RNA was extracted using an Isolate II RNA mini kit. The library was prepared using the TruSeq Stranded mRNA LT Sample Prep Kit, followed by paired-end sequencing (101 bp) on an Illumina platform. Initial sequence quality was assessed using FastQC (v0.11.7), and preprocessing was performed with Trimmomatic (v0.38) to remove adapter sequences and trim bases with a quality score below 3 from the ends. A sliding window approach (window size = 4, mean quality ≥ 15) was used to remove low-quality regions, and reads shorter than 36 bp were discarded, resulting in high-quality, clean data for downstream analysis.