title: GSM4585211: ChIP on POLR2A  and with estradiol treatment of MCF-7 cells (40min) [POL2_shctrl_plusE2_Fan118]; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP was performed as previously described, with minor modifications. Briefly, approximately 5 x 10^7 cells were crosslinked with 1% formaldehyde at room temperature for 15 min and quenched with 0.125M glycine for 5 min. Cells were then harvested and incubated with nuclei preparation buffer (50 mM Tris pH 7.5, 150 mM NaCl , 5 mM EDTA, 0.5% NP-40, 1% TX-100), with protease inhibitor (Sigma, Cat#P2714-1BTL) included, for 10 min. After centrifugation, the pellet was suspended in sonication buffer (10 mM Tris, pH8.0, 100 mM NaCl, 1mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate and 0.5% N-lauroylsarcosine, with protease inhibitor included) for sonication. After sonication, 1/10 volume of 10% Triton X-100 was added to sonicated lysate. The supernatant, after centrifugation, was then incubated with 5μg of antibody at 4°C overnight. Immunoprecipitated complexes were collected using Protein G magnetic beads (Invitrogen, Cat#10004D). Immuno-complexes were washed with RIPA buffer (50 mM Hepes-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40 and 0.7% Na-Deoxycholate, with protease inhibitor included) for at least 5 times and TE buffer containing 50 mM Nacl once. After de-crosslinking, DNA was then purified by QIAquick Spin columns (Qiagen). For Bio-ChIP, 5x10^7 MCF-7 cells stably expressing BirA and biotin acceptor peptide (BAP)-HP1α (4A) or BAP- HP1α (4E) were crosslinked with 1% formaldehyde at room temperature for 15 min, and then incubated with 0.125M glycine for 5 min. To isolate nuclei, cells were incubated with nuclei preparation buffer (50 mM Tris pH 7.5, 150 mM NaCl , 5 mM EDTA, 0.5% NP-40, 1% TX-100), with protease inhibitor (Sigma, Cat#P2714-1BTL) included, for 10 min. The obtained nuclei were suspended in immunoprecipitation buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate and 1% Triton X-100, with protease inhibitor included) for sonication. The sonicated chromatin in immunoprecipitation buffer was then incubated with 50 ul of pre-blocked Pierce™ Streptavidin Magnetic Beads (Thermo Fisher, Cat#88816) overnight at 4°C on a rotating wheel. Beads were extensively washed with SDS buffer (10 mM Tris, pH 8, 1 mM EDTA, 2% SDS, protease inhibitor included) twice, high salt buffer (50 mM HEPES pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate and 500 mM NaCl) twice, DOC buffer (250 mM LiCl, 0.5%, NP-40, 0.5% deoxycholate, 1 mM EDTA and 10 mM Tris pH 8) once and TE buffer containing 50 mM Nacl once. Beads were then treated with RNase A (Thermo Fisher Scientific, Cat# AM2270) and Proteinase K (Thermo Fisher Scientific, Cat# AM2548) before de-crosslinking overnight at 65°C. DNA was finally purified by QIAquick Spin columns (Qiagen, Cat#28106). The library construction protocol followed the manufacturer's instructions.