title: GSM7159354: CHIC-Seq, Pbrm1-WT1; Mus musculus; OTHER
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  CHIC-Seq samples. Small cell number ChIC-seq assays were performed as previous reported. ChIC-seq libraries were constructed using 50,000 mouse primary uterine stromal cells. Cells were fixed with 1% formaldehyde solution at RT for 5 minutes. Reaction was terminated by adding 1:10 volume of 1.25 M glycine. The fixed cells were kept in -80 ℃ after three washes with ice cold PBS. For ChIC reaction and library preparation, cells were incubated and rinsed with 500 ml antibody binding buffer (1xTE pH 7.5+150 mM NaCl+0.1% Trition X100) twice and re-suspended in 100 ml antibody binding buffer. To prepare ProteinA-MNase and antibody complex, Pbrm1 (Abcam,  ab196022), Brg1 (Abcam, ab110641), bounded PA-MNase (Ab+PA-MNase) and were incubated at a molecular ratio of 1:2 on ice for 30 min in 50 ml antibody binding buffer. This complex mixed with 50 ml resuspended cells and incubated for 1.5 h on ice. Cells were washed three times with 1 ml wash buffer (1xTE pH 7.5+400 mM NaCl+1% Triton X-100) and rinsed with 400 ml rinsing buffer (10 mM Tris pH 7.5+10 mM NaCl+0.1% Triton X-100). The MNase digestion was initiated by re-suspending rinsed cells in 40 ml reaction buffer (20 mM Tris, pH 7.4, 10 mM NaCl, 2 mM CaCl2, 0.1% Tx100) and incubated at 37 ℃ for 3 min. The reaction was stopped by adding 80 ml stop buffer (20 mM Tris, pH 8.0, 10 mM EGTA, 20 mM NaCl, 0.2% SDS) and 1 ml proteinase K, then incubated at 65 ℃ for overnight. DNAs were purified by phenol-chloroform extraction. The purified DNA was used for library preparation. For all ChIC-seq libraries, PCR was performed for 13-15 cycles and DNA fragments from 200 bp to 500 bp were isolated and sequenced on NovaSeq 6000 (Illumina).