GSM4403040: scRNA-seq_CTRL; Mus musculus; RNA-Seq
To separate epidermis from dermis, the samples were incubated in HBSS (Gibco) 0,25% trypsin  (Gibco) at 4°C overnight. Epidermis was mechanical separated from the dermis with scalpel first and then by pipetting and trypsin was neutralized by adding DMEM medium (Gibco) supplemented with 2% Chelex Fetal Calf Serum (FCS). Samples were filtrated on 70 and 40µm filter (Falcon). Cells were incubated in 2% FCS/PBS with primary antibodies for 30 min on ice, protected from the light, with shaking every 10 min. Immunostaining for isolating basal IFE and infundibulum cells was performed using FITC-conugated anti-α6-integrin (clone GoH3; BD biosciences). CD34 staining was used to exclude bulge cells from the sorted cells (clone RAM34; BD Biosciences). Primary antibodies were washed with 2% FCS/PBS and cells incubated for 30 min in APC conjugated streptavidin (BD Biosciences), on ice, with shaking every 10 min. Living epidermal cells were gated by forward scatter, side scatter and negative staining for Hoechst dye. Basal cells from the interfollicular epidermis were targeted using CD34 negative and α6 integrin positive gating. Fluorescence-activated cell sorting analysis was performed using FACSAria I at high pressure (70 psi) and FACSDiva software (BD Biosciences). After sorting, 6000 cells were loaded onto each channel of the Chromium Single Cell 3' microfluidic chips (V2-chemistry, PN-120232, 10X Genomics) and individually barcoded with a 10X Chromium controller according to the manufacturer's recommendations (10X Genomics). RNA from the barcoded cells was reverse transcribed, followed by amplification, shearing 5′ adaptor and sample index attachment. The libraries were prepared using the Chromium Single Cell 3' Library Kit (V2-chemistry, PN-120233, 10X Genomics), quantified using a low coverage Illumina NextSeq 550 run and sequenced on an Illumina NovaSeq
GEO
GSM4403040