title: GSM6975310: D2 primary oviductal muscle cell WT1; Mus musculus; ATAC-seq
library_construction_protocol: Oviduct were isolated and cut into small pieces (1-2 mm) and incubated (30-45 min) in rotation at 37 ℃ in pre-warmed DMEM/F12 (1:1) (Gibco), 12 mM HEPES (Gibco), 1% penicillin/streptomycin (Gibco), 2 mg/ml trypsin (Sigma), 400 U/ml collagenase type I (Invitrogen), and 5 U/ml dispase (Invitrogen). The digested cells were passed through a 70-mm filter to obtain a homogenous population of smooth muscle cells. Cells were plated in 100 mm dishes, containing DMEM and Ham F-12 media (1:1) (Gibco) with 12% charcoal-stripped fetal bovine serum (FBS) and antibiotics (penicillin-streptomycin, Thermo Fisher, 100 µg/mL). After 4 h, the medium was replaced with DMEM/F-12 (1:1) containing 10% FBS. 2 h later, the cells were gently washed twice with PBS and samples were prepared for ATAC-seq.  50-100K cell was isolated for ATAC-Seq. ATAC-seq libraries were constructed (Buenrostro et al., 2013) using 50,000 mouse primary oviduct muscle cells. Cells were centrifuged (500 x g, 5 min, 4 °C), washed with 500 ml PBS and re-suspended in 50 ml of cold lysis buffer (10 mM Tris pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.1% digitonin), incubated on ice for 3 min, and diluted with 1 ml wash buffer (10 mM Tris pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20). After centrifugation (500 x g, 10 min, 4 °C), pelleted nuclei were re-suspended in 50 µl of transposition reaction consisting of 2x tagmentation buffer (25 µl), water (5 µl), PBS (16.5 µl), 10% Tween-20 (0.5 µl), 1% digitonin (0.5 µl) and 2.5 ml Tn5 Transposase enzyme (Illumina). After incubation (45 min, 37 °C) with gentle mixing, DNA was purified using a phenol/chloroform extraction kit protocol (Sigma-Aldrich) and eluted with 10 ml of elution buffer. Transposed DNA was amplified using barcoded PCR primers and cycling conditions of 72 °C for 5 min and 98 °C for 30 s followed by 12 cycles of 98 °C for 10 s, 63 °C for 30 s, 72 °C for 1 min. DNA fragments of nucleosome free regions (between primer-dimers and mono-nucleosome bands) corresponding to sequence insert <100 bp or >1000 bp were removed by double-sided beads purification. Libraries were eluted with 15 ml elution buffer for analysis of size distribution and 50 bp paired-end sequencing was provided by the NIDDK Genomic Core Facility.