title: GSM5466893: CD4+Foxp3+ Treg ApoE-/- rep3; Mus musculus; RNA-Seq
library_construction_protocol: Spleen singlet cell suspension were made, stained with anti-mouse CD4 antibody, then CD4+GFP+ cells were purified by Aria Cell Sorter directly into TRIzol, and RNA was harvested using miRNeasy Mini kit. Total RNA libraries were prepared by using Pico Input SMARTer Stranded Total RNA-Seq Kit (Takara). In short, 250 pg -10 ng total RNA from each sample was reverse-transcribed via random priming and reverse transcriptase. Full-length cDNA was obtained with SMART (Switching Mechanism At 5' end of RNA Template) technology. The template-switching reaction was used to keep the strand orientation of the RNA. The ribosomal cDNA was hybridized to mammalian-specific R-Probes and then cleaved by ZapR. Libraries containing Illumina adapter with TruSeq HT indexes were subsequently pooled and loaded to the Hiseq 2500. Single end reads at 75 bp with 30 million reads per sample were generated for bioinformatic analysis.