GSM7500340: Sample_18_WTA; Mus musculus; RNA-Seq
P60 back skin was harvested with surgical scissors and placed dermis-facing up on a styrofoam tray with pins. Fat and subcutis were scraped off with a scalpel. Fat-free skin was then washed, dermis-side facing down in 1xPBS. Skin was then placed in the same orientation in 0.5% Trypsin-EDTA (10X, Gibco; 15400054) and incubated at 37°C on an orbital shaker for 25 (females) or 50 min (males). Using a glass microscopy slide, skin was secured to the bottom of the dish and scraped again with a scalpel until it started to break. Excess cold PBS with 2% chelexed FBS (FBS(-)) was added to neutralize trypsin. Cell suspension was strained on ice first through a 70 µm filter, washed with an additional 15 mL of cold 1X PBS with 2% FBS(-) and then filtered through a 40 µm filter. Cell suspension was spun down at 400 x g for 10 min and resuspended in FACS buffer before sorting mCherry positive cells on a BD Aria II. mCherry-positive cells were sorted on a BD FACSAria using a 70µm nozzle and processed for single-cell capture with a BD Rhapsody Single-Cell Analysis System. For P60 we loaded 8 cartridges (Cat No 633733) with an average of 64625 cells. Reverse transcription from beads and sequencing library production was carried out according to manufacturer's instructions (BD Biosciences, Doc ID: 210967 Rev. 1.0). Whole Transcriptome Amplification Analysis (WTAs) from BD Rhapsody Cartridges were sequenced in full SP flowcells at 1.8nM concentration with 20% PhiX and the following read configuration: Read1 60, I1 8, Read2 62 cycles.