Vps34-intact CD4 T cells 2
FACS sorted cells were pelleted and lysed with RNA lysis buffer from Quick-RNATM MiniPrep (ZYMO Research, Irvine, CA) to extract RNA. RNA quantity and purity were measured with an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA). Total RNA was isolated from biological replicates and provided to the Beijing Genomics Institute for library construction and sequencing following standard protocols. The library products were sequenced using a BGISEQ-500. Briefly, poly-A tailed RNA was purified using paramagnetic oligo-dT beads from 1 g of total RNA. The purified mRNA was chemically fragmented and reverse transcribed to double-strand cDNA using random primers. The adapter-ligated DNA library was amplified and sequenced. Standard bioinformatic analysis was performed by the Beijing Genomics Institute. Briefly, gene expression levels were quantified using RSEM after reads cleaning and genome mapping. Differentially expressed genes (DEGs) were detected with DEseq2, which is based on the negative binomial distribution. DEGs were classified according to official classification with the KEGG annotation result. Pathway enrichment analysis was performed using phyper, a function of R.