GSM8415694: K14-Ctrl, BC, replicate 1; Mus musculus; ATAC-seq
Fresh mamamry glands were mechanically dissected by scissor chopping after draining lymph nodes were removed and enzymatically digested with digestium medium (Dulbecco's modified Eagle's medium (DMEM)/F-12 (Biowest, #L0093-500ml), 0.6 U/ml collagenase A (Sigma-Aldrich, #1108879300), 2.85U/mL dispase II (ThermoFisher Scientific, #17105041), 2% HEPES (ThermoFisher Scientific, #H8357-100ml) and 1% penicillin–streptomycin(ThermoFisher Scientific, #15140-122)) for 1h rocking (50 rpm) at 37°C. Samples were washed with CO2 independent media (ThermoFisher Scientific, # 18045088) supplemented with 2mM glutamax (Gibco, #35050-061). Single cells were isolated by sequential treatments with 0.25%trypsin/0.1% EDTA solution (Sigma-Aldrich, #T3924-100ml) for 1min at RT and dispaseII/DNAseI (ThermoFisher Scientific, #17105041 / Roche, #10104159001) at 37°C for 5 min. Enzymatic digestions were blocked by adding CO2 independent media (ThermoFisher Scientific, #18045088) supplemented with 2mM glutamax (Gibco, #35050-061) and 5% FBS (ThermoFisher Scientific, #F7524-500ml). Erythrocytes were eliminated by treating samples with cold (4°C) ammonium chloride solution (STEMCELL Technologies, #7008) for 1min at RT. Cell aggregates were removed by filtering the cell suspension with a 40μm strainer and single epithelial cells were counted manually using a Neubauer chamber. Single cells were blocked with blocking buffer (PBS + 0.5% Bovine Serum Albumin (Sigma-Aldrich, #13391+ 2mM EDTA) for 20min on ice. Single cells were incubated for 40 min on ice with the corresponding surface antibodies (CD24-AlexaFluor700 (564237, BD Pharmingen™, 1μg/ml), CD49f-AlexaFluor647 (562473, BD Pharmingen™,1.25 μg/ml), CD31-PECy7 (102417, Biolegend, 0.25 μg/ml), and CD45-PECy7 (103113, Biolegend, 0.125 μg/ml)). Prior sorting, single cells solution was filtered by a 70μm strainer. Cell sorting was performed using a BD FACSAriaTM cell sorter.For ATAC-seq, 25000 FACs-isolated cells were washed in cold PBS (500g, 5', 4ºC) and lysed in 50ul lysis buffer (10 mM TrisHCl pH7,5, 10 mM NaCl, 3 mM MgCl2, 0,1% NP40, 0,1% Tween 20, 0,01% Digitonin and 1x Complete Protease Inhibitor Cocktail) for 5 minutes on ice. Nuclei were isolated by centrifugation (500g, 10 min, 4ºC) and treated with transposase Tn5 (, Illumina, Nextera DNA Library Preparation Kit, #FC-131-1024). DNA was purified using MinElute PCR Purification Kit (Qiagen, #28004). Libraries were prepared using NEBNext HF Polymerase with indexing primers from Nextera and sequenced using Illumina Novaseq