GSM7500332: Sample_10_WTA; Mus musculus; RNA-Seq
P4 back skin was surgically removed and scraped for fat, the back skin was washed once in cold PBS and then placed in dispase (Corning; 354235) for 35 min at 37°C, epidermis facing up, on an orbital shaker. Next, epidermis was separated from dermis with fine forceps, torn into smaller pieces and placed in 4 mL 0.25% Trypsin-EDTA (1X, Gibco; 25200056) for 15 min at 37°C with orbital shaking. Epidermis was washed with cold PBS and pipetted vigorously to achieve a single cell suspension. Suspension was then afterwards filtered through a 70 µm and a 40 µm strainer (Corning; 431750, 431751) consecutively, then centrifuged for 10 min at 1400rpm and resuspended in FACS buffer (PBS + 2 % FBS(-) + DAPI) 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 P4 we loaded 8 separate cartridges (Cat No 633733) with an average of 50037 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.