title: GSM3383706: ChIP BHLHB8-HA rep1; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were fixed with 1% formaldehyde for 10 minutes at room temperature and quenched with 200 mM Tris-HCl pH 8.0, washed with PBS, spun down, and stored at -80°C. The cell pellet was resuspended and in 1.5 ml LB1 (50 mM HEPES-KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% Glycerol, 0.5% NP40, 0.25% Triton X-100), incubated 10 min at 4°C, spun down, and resuspended in 1.5 ml LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) incubated 10 min at 4°C. The pellet was spun down, rinsed twice with SDS shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.15% SDS), and finally resuspended in 0.9 ml SDS shearing buffer. All buffers contain contain 1:100 diluted Protease Inhibitor Cocktail in DMSO (Sigma). The suspension was transferred to a milliTUBE 1 ml AFA fiber and sonicated on a E220 focused ultrasonicator (Covaris) using the following settings: 20 min, 200 cycles, 5% duty, 140W, and input sample aliquots were taken. To pull down HA-bound DNA, sonicated chromatin was incubated with anti-HA.11 antibody 16B12 (BioLegend) overnight at 4°C. 2.5 ml Protein G Dynabeads (Thermo Fischer) per one million cells were added to the chromatin and incubated 3 hours at 4°C. The chromatin was washed several times at 4°C with 5 min incubation between each wash and 2 min magnetization to collect beads; twice with Low Salt Wash Buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.15% SDS, 1 mM PMSF), once with High Salt Wash Buffer (10 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.15% SDS, 1 mM PMSF), once with LiCl Wash Buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 250 mM LiCl, 1% NP40, 1% sodium deoxycholate, 1 mM PMSF), and finally with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM PMSF). Beads were finally resuspended in Elution buffer (TE buffer with 1% SDS and 150 mM NaCl), treated with 400 ng/ml Proteinase K and reverse crosslinked at 65°C 1100 rpm overnight. Input samples were treated with 100 mg/ml RNase A and 400 ng/ml Proteinase K and reverse crosslinked at 65°C 1100 rpm overnight. Sonicated chromatin samples were purified using Qiagen MinElute PCR purification kit. Libraries were prepared with NEBNext ChIP-seq Library Prep Master Mix kit with insert size selection of 250 bp.
identifiers.namespace: GEO
identifiers.id: GSM3383706