GSM6475965: mouse muscle ischemia d21 SS2_19_278_K9; Mus musculus; RNA-Seq
Ischemic gastrocnemius muscle was removed and minced into very small pieces with a scalpel. The tissue was then mechanically and enzymatically dissociated by incubating in collagenase II (500 U/ml) (#17101015; Gibco) in RPMI1640 in 37°C for 30 minutes during intermittent pipetting. After washing the tissue with ice-cold DPBS, it was incubated for further 30 minutes with collagenase IV (15 U/ml) (#17104019; Gibco) and dispase (2.4 U/ml) (#17105041; Gibco) in RPMI1640, with intermittent pipetting. Thereafter, the tissue suspension was passed through a 23G 0.6 mm needle 8-10 times, and filtered through a 70 µm cell strainer. Once a single-cell suspension was obtained, live/dead cell staining was carried out using CellTrace Calcein Violet, AM (C34858; Invitrogen), according to manufacturer's instructions. Lastly, tdTomato+PDGFRβ+/- cells and tdTomato-PDGFRβ+ cells were FACS sorted into SMART-Seq2 plates, using BD FACSAria™ III Cell Sorter with 4 lasers configuration (405, 488, 561, 633 nm) (BD Biosciences). RNA libraries were prepared according to Smart-seq2 protocol.