title: GSM7159352: Stromal, ATAC-Seq, Pbrm1 cKO, 3; Mus musculus; ATAC-seq
library_construction_protocol: 5-10 pseudopregnant D4 WT mouse uterine horns were cut into small pieces (2–3 mm). Tissue pieces were first digested in 5 ml fresh medium (HBSS antibiotic; Gibco) containing 6 mg/ml dispase (Gibco) and 25 mg/ml pancreatin (Sigma), and then incubated in fresh medium (3 ml) containing 0.5 mg/ml collagenase (Sigma) at 37°C for 30 min. The digested cells were passed through a 70-lm filter to obtain the stromal cells. Cells were plated at 60-mm dishes, containing DMEM and Ham F-12 nutrient mixture (1:1) (Gibco) with 10% charcoal-stripped fetal bovine serum (FBS) and antibiotic. After 4 h, the medium was replaced with fresh medium (DMEM/F-12, 1:1) with 10% FBS. 2 hours later, wash twice with PBS gently for the preparation of ATAC-Seq samples. ATAC-seq libraries were constructed (Buenrostro et al., 2013) using 50,000 mouse primary uterine stromal 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.