title: GSM7113241: BackSkin Wildtype with CellPlex library; Mus musculus; RNA-Seq
library_construction_protocol: To isolate skin epidermal cells, ears were dissected, opened in two along the cartilage with forceps and incubated in DMEM (Dubelcco's Modified Eagle's Medium- Gibco, thermo Fisher Scientific) - 0,25% Trypsin (Gibco, Thermo Fisher Scientific) overnight at 4°C. The back skin was first shaved with electric clippers, dissected and the hypodermis was then removed by scratching it with a scalpel. The remaining tissue (epidermis-dermis) was finally incubated in DMEM-0,25% trypsin overnight at 4°C by placing the dermis in contact with the solution. The day after, the epidermis was carefully separated from the dermis by using a scalpel and then incubated on a rocking plate (100 rpm) at room temperature for 5 min. Trypsin was neutralized by adding DMEM supplemented with 5% of Chelex-treated Fetal Calf Serum (CFCS) and epidermal cells were mechanically dissociated by pipetting 10 times. Samples were filtrated on a 70µm filter then on a 40µm filter and washed in a PBS-2% CFCS. Single cells were incubated with a biotinylated anti-CD34 primary antibody (clone RAM34; BD Biosciences; dilution 1:50) diluted in PBS-2% CFSC, for one hour on ice on a rocking plate, and protected from light. Cells were washed with PBS-2% CFCS, incubated for 45 min on ice, on a rocking plate, with streptavidin-APC secondary antibody (BD Biosciences; 1:400), washed again and finally resuspended in a Hoechst (10mg/ml) solution diluted at 1:4000 in PBS-2% CFCS.  Cell sorting was performed on a BD FACS Aria II using the FACSDiva software at the ULB Flow Cytometry Platform: living epidermal cells were gated by forward scatter, side scatter, and negative for Hoechst. SmoM2 mutated cells were isolated based on the expression of the YFP fused to the oncogene. Smom2-YFP expressing cells from the hair follicle bulge were excluded by the CD34-APC staining.  For wild type tissues living IFE and infundibulum cells were sorted on the negative expression of the CD34 marker only. 10,000 single cells from the back skin and ear skin were loaded onto each channel of the Chromium Single Cell 3′ microfluidic chips (V2-chemistry, PN-120232, 10X Genomics) and barcoded with a 10X Chromium controller according to the manufacturer's recommendations (10X Genomics). RNA from the barcoded cells was subsequently reverse transcribed, followed by amplification, shearing 5′ adaptor and sample index attachment. The libraries were prepared using the Chromium Single Cell 3′ Library Kit (V3-chemistry, PN-120233, 10X Genomics) and sequenced on an Illumina Novaseq 6000 (paired-end 100bp reads).