GSM7109889: Macrophages, cKO, replicate 2; Mus musculus; ATAC-seq
Back skins from Igf1-flox and Csf1r-Cre x Igf1-flox female adult mice were harvested after shaving with an electric clipper. Subcutaneous fat was removed from hypodermis and hypodermal layers were physically isolated from the dermis with forceps. Hypodermis was minced with scissors and digested for 2H at 37°C in RPMI supplemented with 5% FCS and containing 0.25mg/mL of liberase T-Flex and 1ug/mL of DNAse I. Cells were isolated and immunostained with anti-CD45 and anti-CD64 antibodies as well as stained with Sytox-Red (SR) before being FACS sorted as live macrophages (SR–, CD45+, CD64+). Cells were lysed in cold lysis buffer (10 mM Tris–HCl (PH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonidet P40, 0.01% Digitonin, 1mM DTT and 1U/μl RNase inhibitor) for 5 min on ice for nuclei isolation. Upon washing the nuclei extracts, targeted nuclei recovery was processed for single-cell ATAC-sequencing. Library construction was performed in accordance with 10X Genomics protocol (Next GEM Single Cell Multiome ATAC + Gene expression (GEX) protocol, CGOOO338).