GSM8603395: WT-MP-rep1; Mus musculus; miRNA-Seq
Adult or neonatal spleens were isolated in complete media and CD8+ T cells were positively selected for using magnetic microbead (Miltenyi) selection according to manufacturer's instructions. Cells were then prepped for naive CD8 subset FACS sorting on the SONY by staining with CD8, CD4, CD44 and CD122 and Viability Dye (∼97% purity) and 200,000+ CD8+ cells sorted into PBS + 2% BSA + 2 mM EDTA. 10,000 FACS sorted cells were placed in Trizol and RNA was extracted according to manufacturer's instructions, with the addition of a chloroform extraction, Glycoblue (ThermoFisher) carrier prior to precipitation (1 hour at 4°C), and a second wash of the pelleted RNA in 70% ethanol. RNA integrity was confirmed on a Fragment Analyzer (AATI). Libraries were prepared with NEBNext Small RNA Library Prep Kit (New England Biolabs) using 40-50ng total RNA as input, size selected for insert sizes ~18-35bp. small RNA-seq