GSM4986910: Ms1537-Normal; Mus musculus; RNA-Seq
3 weeks after the MI surgery, mice were euthanized and perfused with PBS supplemented with EDTA, and hearts were harvested. Infarcted tissue was dissected under a stereomicroscope, and digested with DMEM medium with collagenase-II (5 mg/ml, Invitrogen 17101-015) and DNase (1 mg/ml, Sigma D4527). Single cell suspension was prepared after filtering with a mesh strainer (100-micron pores). Cells were incubated with anti-F4/80 antibody (Miltenyl Biotec, #130-102-327), and tdTomato+F4/80- cells were sorted by using a flow cytometer (BD FACS Aria). RNA was extracted using TRIzol (Thermo Fisher, 15596026) according to the manufacturer's instructions, followed by RNA purification using a RNeasy Plus Mini Kit (Qiagen). cDNA was obtained by reverse transcription with Superscript III (Thermo Fisher, 18080044), and a second complementary cDNA strand was synthesized by DNA polymerase (10 U/ul, Thermo Fisher, 18010025), followed by in vitro RNA transcription using a MEGASCRIPT T7 KIT (Thermo Fisher, AMB13345) and RNA purification using a MEGACLEAR KIT (Thermo Fisher, AM1908). Quality control was validated using a RNA Nano assay chips with a 2100 Bioanalyzer (Agilent) The library was constructed according to a TruSeq protocol (Agilent), followed by sequencing (100 PE, about 10 megabyte reads for each sample) with a 2500 Illumina sequencer at the Children's Hospital of Philadelphia/Beijing Genomics Institute core facility (CHOP/BGI).