title: Illumina HiSeq 2500 paired end sequencing; RNAseq of bulk regulatory T cells (Tregs) and conventional T cells (Tconvs) during acute kidney injury and fibrosis development
design: RNAseq of bulk regulatory T cells (Tregs) and conventional T cells (Tconvs) during acute kidney injury and fibrosis development
library_construction_protocol: For Treg bulk RNAseq, 3-5 biological replicates of 3000-5000 Treg or Tconv cells, consisting of a pool of 5 to 10 mice, were FACS sorted in lysis buffer (Qiagen) containing 1% of 2-Mercaptoethanol (Sigma) and preserved at -80°C until RNA extraction. The Tregs were sorted as live-singlets CD45+CD4+TCRB+ Foxp3-GFP+ and Tconv were isolated as live-singlets CD45+CD4+TCRB+Foxp3-GFP-. In all cases, purity exceeded 90%. RNA was extracted using RNeasy micro kit (Qiagen), according to manufacturer's instructions. cDNA was synthesized using high capacity RNA to cDNA kit (Applied Biosystems) Quality and quantity of RNA was tested with the Agilent 2100 Bioanalyzer using the RNA 6000 Pico Chip and reagents (Agilent).  RNA-seq libraries were prepared with Illumina TruSeq Stranded total RNA Library Prep kit with RiboZero Gold according to manufacturer's instructions.