title: GSM8127352: WT, ATACseq Rep2; Mus musculus; ATAC-seq
library_construction_protocol: Thoracic and inguinal mammary glands were resected, lymph nodes removed, and samples collected into cold MEC media (DME-F12 supplemented with 1 mM glutamine, 5µg/ml insulin, 500ng/mL hydrocortisone, 10ng/mL Epidermal growth factor, 5% FBS). Glands were mechanically dissociated with a McIlwain tissue chopper then digested for 1 hour at 37°C in an orbital shaker with 300 U/mL collagenase, 100 U/mL hyaluronidase and 0.1 mg/mL DNAse I in MEC media containing 5% FBS. The resulting suspension was diluted in PBS with 2% FBS and cells pelleted by centrifugation. The cell pellet was further digested in 0.25% trypsin-EGTA with 0.1 mg/mL DNAse I for 3 minutes at 37°C, then diluted in PBS with 10% FBS and cells pelleted by centrifugation. The cell pellet was then digested in 5mg/mL Dispase with 0.1mg/mL DNAseI at 37°C for 5 minutes, then diluted in PBS with 2% FBS. Cells were passed through a 100 mm cell strainer to obtain a single cell suspension, then live cells counted using trypan blue. Single cells were pelleted by centrifugation, then blocked in rat γ-globulin and anti-CD16/CD32 Fcγ III/II receptor antibody with 0.1mg/mL DNAseI for 10 minutes at RT. Antibody incubations were performed at 4°C for 25 minutes. Antibodies used for Fluorescence-activated cell sorting (FACS) were: APC anti-mouse CD31, APC anti-mouse CD45 Antibody, APC anti-mouse TER-119/Erythroid Cells, Pacific Blue anti-mouse CD24 and FITC-anti-mouse CD29. Cells were diluted in PBS with 2% FBS then pelleted by centrifugation, and 0.2mg/ml 7-Aminoactinomycin D added to exclude dead cells. The cell suspension was then filtered through a 40 µm cell strainer and luminal mammary epithelial cells (Ter119–CD31−CD45–CD24+CD29Lo) sorted on a FACS Aria using a 100 µm nozzle. Nuclei were extracted from 50,000 cells per sample by resuspending in 50 µl of ATAC-seq lysis buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% IGEPAL, 0.01% Digitonin) and incubating on ice for 3 minutes followed by washing with   10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20. Samples were then centrifuged at 500 g for 10 minutes at 4°C and the supernatant discarded.  Transposition of the DNA and ligation of adapters was performed using the Illumina Tagment DNA TDE1 Enzyme and buffer kit (20034197) as per the manufacturer's instructions. Briefly, DNA was digested by incubation with Tn5 Transposase enzyme at 37°C for 30 minutes. Transposed DNA was then purified using the Qiagen MinElute reaction cleanup kit (Qiagen, 28204), and partially PCR-amplified using Nextera custom barcoded primers for 5 cycles. To reduce GC-bias, the number of additional PCR cycles required was determined to be 9 cycles (14 cycles in total) by using a SYBR Green q-RT-PCR reaction and calculating the number of cycles corresponding to 1/3 of the maximum fluorescent intensity. Libraries were then purified by AMPure XP magnetic bead purification (Beckman Coulter A63880). DNA quantity and quality was assessed using the Tapestation 2000 system (Agilent).