RNA-Seq of CD45-CD31+ ECs from ischemic muscles of Lepr db/db and Lepr db/+ mice.

Published: 12 June 2018| Version 1 | DOI: 10.17632/4n2whfwvd6.1
Contributor:
Kevin YANG

Description

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.

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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).

Institutions

Chinese University of Hong Kong Faculty of Medicine

Categories

RNA Sequencing

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