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

Published: 12 Jun 2018 | Version 1 | DOI: 10.17632/4n2whfwvd6.1
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Description of this data

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.

Experiment data files

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).

Latest version

  • Version 1

    2018-06-12

    Published: 2018-06-12

    DOI: 10.17632/4n2whfwvd6.1

    Cite this dataset

    YANG, Kevin (2018), “RNA-Seq of CD45-CD31+ ECs from ischemic muscles of Lepr db/db and Lepr db/+ mice.”, Mendeley Data, v1 http://dx.doi.org/10.17632/4n2whfwvd6.1

Institutions

Chinese University of Hong Kong Faculty of Medicine

Categories

RNA Sequencing

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The files associated with this dataset are licensed under a Creative Commons Attribution 4.0 International licence.

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