title: GSM6460378: infected_Cacnes42_mouse_scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: All animal experiments were approved by the University of California, San Diego (UCSD), Institutional Animal Care and Use Committee (#S09074). For single cell RNA-Seq analysis of mouse skin, the upper back skin of SKH-1 mice were intradermally infected with 1 x 107 CFU of C. acnes or RCM control for 3 days. Skin samples were collected from 6 mm punch biopsies and immediately placed on ice. In total, 3 biopsies were pooled per condition (infected or non-infected) for single cell generation. Single cell suspensions were generated by cutting skin into small pieces using a scalpel followed by digestion in Hanks Balanced Salt Solution (HBSS) (ThermoFisher, #14175095) supplemented with 20 mg/ml Bovine Serum Albumin (BSA), 1X Antibiotic-Antimycotic (ThermoFisher, #15240062), 50 U/ml DNAse I (Sigma, #04716728001), 0.1 mg/ml Liberase (Sigma, #05401020001), 20 mM HEPES (ThermoFisher, #15630080), 2 mM sodium pyruvate (ThermoFisher, #11360070) and 1 mg/ml Collagenase Type IV (ThermoFisher, 17104019). Tissue dissociation was carried out by a 2 h ice-cold incubation in the digestion buffer followed by 45 min incubation at 37°C shaking at 200 rpm. Enzymatic dissociation was terminated by addition of 5 mM EDTA and 10% Fetal Bovine Serum (FBS). Digests were filtered twice through a 40 μm filter, then incubated in red blood cell lysis buffer followed by removal of dead cell using MACS Dead Cell Removal Kit (Milteny Biotek) for downstream applications in 10X Genomics Chromium platform. Single cell suspensions were loaded onto the 10X Genomics Chromium Controller instrument to generate single cell GEMs. GEM-RT and library construction were performed following the 10X Genomics Protocol.