GSM8603388: Dirty_TN_rep1; Mus musculus; miRNA-Seq
Splenic CD8+ T cells were isolated and purified using positive selection with CD8a microbeads (Miltenyi). Purified CD8+ T cells were stained using antibodies against CD4 (Gk1.5), CD44 (IM7), CD122 (TM-B1), CD49d (R1-2), and CD8a (53-6.7). 'Stamped VMs' populations were sorted for CD4-CD8+CD49dloGFP+CD44hi, and 'stamped TN' populations were sorted for CD4- CD8+CD49dloGFP+CD44lo. Samples were sorted using a BD FACS Aria Fusion II equipped with four lasers. Samples were sorted to >95% cell purity as determined by a post-sort check. 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 60-100ng total RNA as input, size selected for insert sizes ~18-35bp. small RNA-seq