GSM4976296: ZR751shMLL3_ERa_rep2; Homo sapiens; ChIP-Seq
Cells were harvested at steady-state using the RNAEasy Kit (Qiagen 74104). ChIPs were performed for two biological replicates, for one experimental repetition. Cells were grown to 80% confluency, washed 3 times in ice-cold PBS (8g NaCl, 0.2g KCl, 1.44g Na2HP04, 0.24gKH2PO4, H2O up to 1L, adjusted to pH 7.4 with HCl) and then fixed for 10 minutes at room temperature using 7% formaldehyde (50ml cold PBS, 2.5ml formaldehyde Sigma Aldrich 252549), followed by quenching with 2.5 ml of 2.5M glycine (93.8g glycine Sigma Aldrich G7126 in 500ml H2O) for two minutes at room temperature. After aspirating and washing with 50 ml cold PBS, we lysed the cells using 20ml Farnham lysis buffer (5mM HEPES pH 8.0, 85mM KCl, 0.5% NP-40) and 400ul protease inhibitor cocktail (PIC, Roche 11873580001) in order to scrape the cells off (Corning™ 3008) into a 50ml conical tube (Corning 352098). These tubes were spun down at 2,000 rpm for five minutes at 4° Celsius.     Nuclei lysis buffer (50mM Tris-HCl ph8, 10mM EDTA pH8, 1% SDS), 1X PIC, and 10mM sodium butyrate (Sigma Aldrich B5887) were added to a concentration of ~2e7 cells per 400 ul and resuspended until homogenous. Chromatin was sonicated using a Covaris LE220 for 35 minutes, then centrifuged at max speed for 10 minutes at 4°C to obtain supernatant. Per 0.1 ml of supernatant, we diluted with 0.9ml ChIP dilution buffer (50mM Tris-HCl pH 8, 0.167M NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate), 0.5ml RIPA-150 (50mM Tris-HCl pH8, 0.15M NaCl, 1mM EDTA pH8, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), 28ul 50X PIC, and 14ul 1M sodium butyrate.      Anti-ERα (3ul/IP), anti-H3K4me1 (1ul/IP), and anti-SP1 (3ul/IP) were linked to 100ul/IP, 60ul/IP, and 100ul/IP magnetic anti-rabbit Dynabeads respectively with RIPA-150 up to 500ul for 6 hours at 4°C for 6 hours in low-bind tubes (Eppendorf Z666505), and then incubated with 150ug of chromatin overnight at 4°C. Immunoprecipitants were washed with RIPA-150, RIPA-500 (50mM Tris-HCl pH8, 0.5M NaCl, 1mM EDTA pH8, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate) twice, RIPA-LiCl (50mM Tris-HCl pH8, 1mM EDTA pH8, 1% Nonidet P-40, 0.7% sodium deoxycholate, 0.5M LiCl2) twice, and 1X TE Buffer pH8 (10mM Tris-HCl pH8, 1mM EDTA pH8) twice for 5 minutes each. Chromatin-IPs were eluted from the beads in 200 ul freshly made Direct Elution Buffer (10mM Tris-HCl pH8, 0.3M NaCl, 5mM EDTA pH8, 0.5% SDS), and then treated with 1ul 1mg/ml RNase A (Fisher Scientific FEREN0531) at 65°C with shaking for 4 hours to reverse crosslinks. This was followed by 3ul proteinase-K (Sigma-Aldrich 3115879001) overnight at 55°C. DNA was purified using phenol–chloroform extraction. Samples were transferred to a spun-down 2ml phase lock gel tube (Qiagen 129056) and an equal volume of phenol/chloroform/isoamyl alcohol (Sigma Aldrich P3803100ML) was added. This was spun at room temperature for 5 minutes at 14,000g, and the sample was moved to a new 1.5ml tube.  One tenth volume sodium acetate (Invitrogen AM9740), 1ul glycogen (Roche 10901393001), twice volume 100% ethanol (Sigma Aldrich E7023500ML) was added, and the samples were incubated at -80°C for 30 minutes. The sample was spun at max speed for 30 minutes at 4°C, and the supernatant was carefully aspirated. The pellet was washed with 1ml cold 70% ethanol, and spun at max speed for 30 minutes at 4°C. The supernatant was aspirated, and the spin was repeated a final time. The supernatant was removed, and pellet was allowed to dry. The pellet was then resuspended in 25ul elution buffer (Qiagen 19086) and subsequently quantified by Qubit 2.0 Fluorometer. Standard Illumina ChIP-seq Library Kits (IP-202-1012, IP-202-1024) were used to build sequencing libraries for two biological replicates per condition for one experimental repetition, with inputs used as control. . RNA quality was assessed in VANTAGE via Invitrogen Qubit and Agilent BioAnalyzer and samples with RIN >7 were used. RNA libraries were generated with two biological replicates of 2 μg RNA using Illumina's TruSeq Stranded Total RNA Sample Prep Kit (20020597).