GSM1973010: Effector_RNA-Seq; Mus musculus; RNA-Seq
On day 8 post-infection (p.i.), CD45.2+CD8+KLRG1+ IL-7Ra– cells were sort-purified as terminally differentiated effector CD8+ T cells. On ≥ day 60 p.i., CD45.2+CD8+CD62L+ cells were purified as central memory CD8+ T cells. From the lymph nodes of uninfected P14 TCR-Tg mice, CD62L+CD44lo-med CD8+ cells were isolated as naïve CD8+ T cells. The cell sorting was performed on on FACSAria (BD Biosciences). Total RNA was isolated from sorted CD8+ T cells. RNA Integrity was assessed using RNA 6000 Pico Kit (5067-1513) for Bioanalyzer 2100 (Agilent). mRNA was isolated from total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490, NEB). PolyA-selected mRNA was fragmented to an average size of 300 nt, reverse transcribed to generate double-stranded cDNA, and converted to a paired end library using NEBNext Ultra RNA Library Prep Kit for Illumina (E7530, NEB) according to manufacturer's instructions including the optional double size selection procedure using Agencourt AMPure XP beads. Prepared libraries were quantified with dsDNA HS Kit (Q32851) for Qubit and the size distribution was assessed using High Sensitivity DNA Kit (5067-462) for Bioanalyzer.