GSM4654445: YFP+ Memory Rep1; Mus musculus; RNA-Seq
CD4+ cells were purified using LS positive selection magnetic-activated columns (MACS) and anti-CD4 (L3T4) beads (Miltenyi Biotec). YFP- and YFP+ Naive and Memory CD4+ MACs sorted cells were further purified by fluorescence activated cell sorting using a FACS Aria to purify Naïve (CD4+ CD25- CD44low CD62Lhigh) or memory (CD4+ CD25- CD44high CD62Llow) cells based on the basis of YFP expression. RNA was extracted using the RNAeasy Micro kit (Qiagen) and RNA samples were then checked for the quality, contamination and concentration using a NanoDrop, Qubit spectrophotometer and Agilent Bioanalyzer. Libraries were prepared using the Ovation SoLo RNA-seq library preparation kit (NuGEN genomics) according to the manufacturer's protocol and were sequenced on an Illumina HiSeq 3000 to generate 2x75 bp paired-end reads.