title: GSM3929437: ChIPseq_nodox_rep2; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP-seq: Day 4 EBs were prepared for ChIP from doxycycline treated (24 hrs) and untreated (control) conditions. Day 4 EBs were dissociated with trypsin/EDTA solutionvat 37°C for 1.5 min with gentle shaking and reaction was inhibited by adding 10% FBS/PBS. Washed cells were treated with 1% formaldehyde (Pierce) to crosslink protein-DNA complexes (10 min at RT) followed by quenching with glycine. Cell pellets were incubated sequentially in lysis buffers 1, 2, 3 supplemented with protease inhibitors (complete-mini protease inhibitor cocktail (Roche)) for 10 min each at 4°C (LB1: 50mM HEPES KOH, pH7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100; LB2: 10mM Tris-HCl, pH 8, 200mM NaCl, 1mM EDTA, 0.5 mM EGTA; LB3: 10mM Tris-HCl, pH 8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine) and then sonicated for four cycles with a probe tip sonicator at 18% power for 1 min with intervals of 10 s ON-10 s OFF to reach an average chromatin size of 300bp. After shearing, samples were centrifuged for 10 min at 16,000g and snap-frozen in liquid nitrogen if not processed immediately. For each ChIP, 25mg of chromatin was precleared for 4 hr at 4°C with 15µl of BSA-blocked Protein A-conjugated sepharose beads (GE healthcare) and then supplemented with 1/10 volume of 10% Triton X-100. Immunoprecipitation was performed by overnight incubation with normal mouse IgG (5µg - Millipore) or anti-myc antibody (5µg clone 9E10 – Roche). Immune complexes were recovered by incubation with 15µl of BSA-blocked Protein A-conjugated sepharose beads for 4 hr at 4°C and then washed five times with RIPA wash buffer (50mM HEPES KOH, pH7.5, 500mM LiCl, 1 mM EDTA, 1% NP40, 0.25% Triton X-100 and 0.7% sodium deoxycholate) and one time with TEN buffer (10mM Tris-HCl, pH 8, 1mM EDTA and 50mM NaCl). Immunoprecipitated chromatin was recovered by incubating beads with 200µl of elution buffer (50mM Tris-HCl, pH 8, 10mM EDTA and 1% SDS) for 20 min at 65°C. Chromatin from immunoprecipitation and input (equivalent to 1% of starting material) was reverse crosslinked overnight at 65°C, then diluted 1:1 with TE (10mM Tris-HCl, pH 8, and 1mM EDTA) supplemented with 4µl of RNaseA (20mg/ml – ThermoFisher) and incubated for 2 hr at 37°C followed by Proteinase K treatment (4µl of 20mg/ml) for 30 min at 55°C. DNA was purified using Phenol-chloroform-isoamyl alcohol extraction, precipitated, washed, and dissolved in 50µl H2O. ChIP-seq libraries were generated using the NEBNext DNA library prep kit (NEB) and AMPure XP beads (Beckman Coulter) for all the purification and size selection steps. 10ng or less of DNA were processed following manufacturer's instructions. ATAC-seq: 50,000 freshly sorted cells from differentiated ES cultures were washed with 200 µl of cold PBS then resuspended in 100 µl of cold lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-630), spun at 500 g for 10 minutes at 4°C and resuspended in 50 µl of the transposition reaction mix. Transposition occurred at 37°C for 30 minutes, after which transposed DNA was purified using a Qiagen MinElute Kit and eluted in 10µl Elution Buffer. Final libraries were generated by primer extension of the transposed DNA followed by PCR amplification using Illumina-compatible adapter-barcodes. RNA-seq: d4 EBs were collected and RNA was prepared using the Purelink RNA mini kit (ThermoFisher). RNA-seq libraries were generated using the TruSeq stranded kit and dual-index adapter-barcodes following manufacturer's instructions.
identifiers.namespace: GEO
identifiers.id: GSM3929437