RNA-Seq of CD45-CD31+ ECs from ischemic muscles of Lepr db/db and Lepr db/+ mice.
To delineate the genotypic difference in ECs of Leprdb/+ and Leprdb/db, we purified CD45-CD31+ ECs from the ischemic muscles by flow cytometry; and performed genome-wide transcriptomic profiling by RNA-Seq.
Steps to reproduce
For mouse tissues, the dissociated mixture of muscle cells, ECs and immune infiltrates were first incubated with 2% rat serum, followed by staining with rat fluorochrome-conjugated antibodies against the following mouse antigens: CD31, CD45 (BD Biosciences or Biolegend) at 4°C for 30 minutes. Cells were then washed three times with 2% FBS-containing PBS and analyzed on flow cytometer. Total RNA was isolated and analyzed on Agilent Tape station for RNA Integrity Numbers (RIN) prior to library preparation. RNA-Seq libraries were prepared using TruSeq Stranded mRNA Library Prep Kit according to manufacturer’s protocol (Illumina). mRNA was isolated using poly-T oligos conjugated to magnetic beads, and then fragmented and reverse-transcribed to cDNA. dUTPs were incorporated during second strand synthesis and thus not amplified. cDNA underwent end-repair, ligation with indexed adapters and PCR amplification. Nucleic acid was cleaned up after each steps using AMPure XP beads (Beckman Coulter). Libraries were quantified, pooled and sequenced at single-end 50 base-pair on the Illumina HiSeq platform. Raw sequencing data was demultiplexed and converted into FASTQ files using VASAVA (v1.8.2).