GSM4977800: Uninjured; Mus musculus; RNA-Seq
Mouse quadriceps were extracted, and separately weighed using sterile surgical tools and placed into separate petri dishes containing ice-cold PBS. Using surgical scissors, muscle tissues were minced and collagenase type II (Gibco 17101015, 0.2%) and dispase II (Sigma D4693, 2.5U/mL) were added to 10mL of DMEM (Gibco-Invitrogen Cat#10569010) per mouse. Samples were placed on rocker in a 37˚C incubator for 1 hour and mixed by pipette every 30 minutes. The enzymes within the slurry were then inactivated by addition of 20% heat-inactivated fetal bovine serum (HI-FBS) in Ham's F10 media (Gibco-Invitrogen # 11550-043). The solution was passed through a 70um cell strainers, centrifuged, washed, and counted. Cells were then divided for fluorescent activated cell sorting (FACS) enrichment of viable cells and removal of debris Freshly isolated cells were briefly stained with Propidium Iodide (PI)and FACS sorted to remove dead cells (PI positive) and debris. 10,000-16,000 cells were loaded into the 10x Genomics chromium single cell controller and single cells were captured into nanoliter-scale gel bead-in-emulsions (GEMs). cDNAs were prepared using the single cell 3′ Protocol as per manufacturer's instructions and sequenced on a NovaSeq 6000 flow cell (Illumina) with 26 bases for read1 and 98x8 bases for read2.