GSM5625410: ChIP_BioSmad7_rep1; Mus musculus; ChIP-Seq
4×107 ESCs expressed Bio-Smad7 protein were harvested, cross-linked with 1% formaldehyde for 10 min, quenched with 0.125 M glycine for 5 min, and then washed twice in cold 1×PBS. Cells were lysed in Isotonic Buffer (20 mM HEPES, 100 mM NaCl, 250 mM Sucrose, 5 mM MgCl2, 5 µM ZnCl2, pH 7.5) supplemented with 1% NP-40 and anti-protease. Cell lysates were centrifuged at 1,000 rpm for 5 min at 4°C to isolate the nuclei. Nuclei were suspended in 1 mL of 0.15 % SDS Lysis Buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 0.15 % SDS, 1 % Triton X-100, pH 8.0) supplemented with anti-protease and subjected for sonication to shear chromatin fragments to an average size between 100bp and 400bp on the Pico Bioruptor® (Diagenode) (2 runs of 10 cycles [30 sec ''ON'', 30 sec ''OFF'']) at high power setting. Fragmented chromatin was centrifuged at 13,000 rpm for 15 min at 4°C.  50 µL MyOneTM Streptavidin T1 Dynabeads (Thermo-Fisher Scientific) were saturated with PBS/1% BSA at RT for 1 hour and then incubated with the sample at 4°C overnight on a rotator. After incubation, Dynabeads were washed twice with 1 mL of Washing Buffer I (2 % SDS), once with 1 mL of Washing Buffer II (50 mM HEPES, 500 mM NaCl, 0.1% Sodium Deoxycholate, 1% Triton X-100, 1 mM EDTA, pH 7.5), once with 1 mL Washing Buffer III (10 mM Tris-HCl, 250 mM LiCl, 0.5% NP-40, 0.5% Sodium Deoxycholate, 1 mM EDTA, pH 8.0), and once with 1 mL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). All the washing steps were performed at RT for 8 mins on a rotator. The chromatin was eluted in 250 µL SDS Elution Buffer (1% SDS, 50 mM Tris-HCl, 10 mM EDTA, pH 8.0) followed by reverse cross-linking at 65°C overnight. After decrosslinking, the ChIP DNA was purified using QIAQuick PCR Purification Kit (Qiagen) according to the manufacturer's instructions. For the reference sample, BirA-ESCs without tagged protein were used. The purified ChIP DNA was processed for library generation using the NEBNext ChIP-seq Library Prep Master Mix (NEB) following the manufacturer's protocol. Libraries were pooled and sequenced on an Illumina Next500 Platforms using the 75bp high output sequencing kit.