title: GSM7830682: H3K27ac_Rln_replicate1; Mus musculus; ChIP-Seq
library_construction_protocol: Pools of frozen myometrial tissue (5 myometrium per pool; 60 to 80 mg of tissue) were powdered using a metal pulverizer.  The powdered myometrial pool was immediately resuspended in 1% formaldehyde in phosphate-buffered saline (PBS) with a volume of 10 mL per pool, then incubated on a rotating platform at room temperature for 10 min.  Crosslinking was quenched by adding 5 mL per pool of 2.5 M glycine, with a subsequent 5 min incubation at room temperature.  The crosslinked tissue was collected by centrifugation at 1000 x g for 3 min at room temperature.  The supernatant was decanted, and the tissue was resuspended in 4°C PBS with added protease inhibitor cocktail (Roche Molecular Biochemicals) at 10 mL per pool, with incubation on ice for 2 min.  The samples were centrifuged at 1000 x g for 3 min at 4°C.  The PBS wash was repeated, and the tissue was resuspended in 2.5 mL per pool of homogenization buffer (50 mM Tris-HCl pH 7.5, 1% Nonidet P-40, 0.25% deoxycholic acid, 1 mM EDTA) supplemented with proteinase inhibitor cocktail. The crosslinked tissue was homogenized using a cooled blade homogenizer (Power Gen25).  The pellet was collected by centrifugation at 1000 x g for 5 min at 4°C.  The supernatant was decanted and the pellet was resuspended in 1 mL per pool of Farnham Buffer (5 mM PIPES, pH 8.0, 85 mM KCl, 0.5% Nonidet P-40, 1 mM DTT, 0.1 mM PMSF) supplemented with proteinase inhibitor cocktail by mixing five times with a pipettor and then incubated on ice for 10 min.  The chromatin was pelleted by centrifugation for 1000 x g for 10 min at 4°C and resuspended in SDS Lysis Buffer (50 mM Tris-HCl pH 7.9, 10 mM EDTA, 1% SDS, 1 mM DTT) supplemented with proteinase inhibitor cocktail and histone deacetylases inhibitors (10 mM sodium butyrate, 5 mM nicotinamide).  Resuspended chromatin was incubated on ice for 10 min, split into 300 mL aliquots, and sonicated on ice using a Bioruptor UCD-200 (Diagenode) on the high setting for 9 cycles, 30 sec on and 30 sec off with 1 min rest, 4 times, to obtain genomic DNA fragments of approximately 200 to 400 bp, as determined by gel electrophoresis.  Sonicated chromatin was centrifuged at maximum speed in a microcentrifuge for 1 min at 4°C to remove insoluble debris, and the soluble chromatin supernatant was flash frozen in liquid N2 and stored at -80°C until use. Chromatin was diluted 1:9 with Dilution Buffer (20 mM Tris-HCl pH 7.9, 300 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 1 mM DTT, 0.2 mM PMSF) supplemented with proteinase inhibitor cocktail and histone deacetylases inhibitors.  The diluted chromatin was pre-cleared with 20 mL/mL protein A-agarose beads (ThermoFisher Scientific; 50% slurry in 10 mM Tris-HCl pH 8.1; 1 mM EDTA) on a nutator for 1 hour at 4°C.  The pre-cleared chromatin was centrifuged in a microcentrifuge at 1000 x g for 3 min at 4°C.  The pre-cleared supernatant was divided into input (2.5%) and a 1 mL aliquot for immunoprecipitation.  Immunoprecipitation was performed overnight at 4°C using the polyclonal antibodies for ERa, H3K27ac, or H3K4me1.  For a control, rabbit IgG (BioRad; PRABP01) was added at 1:200. Immune complexes were collected by the addition of 80 mL of protein A-agarose beads (Cell Signaling Technology; 9863) with an additional 2 hours incubation at 4°C with mixing.  The samples were centrifuged in a microcentrifuge at 500 x g at 4°C for 1 min to collect the resin.  The resin was washed four times for 10 min each in Low Salt Wash Buffer (20 mM Tris-HCl pH 7.9, 2 mM EDTA, 125 mM NaCl, 0.05% SDS, 1% Triton X-100), four times for 10 min each  in High Salt Wash Buffer (20 mM Tris-HCl pH 7.9, 2 mM EDTA, 500 mM NaCl, 0.05% SDS, 1% Triton X-100], one time with LiCl Wash Buffer (10 mM Tris-HCl pH 7.9, 1 mM EDTA, 250 mM LiCl, 1% Nonidet P-40, 1% sodium deoxycholate), and one time in TE.  All buffers were supplemented with proteinase inhibitor cocktail and histone deacetylases inhibitors.  The cross-linked chromatin was then eluted twice using Elution Buffer (1% SDS and 100 mM sodium bicarbonate) at room temperature for 15 min.  The eluted chromatin was heated at 65°C overnight in Elution Buffer supplemented with 100 mM NaCl to reverse the formaldehyde crosslinking.  RNase was added to 3 mg/mL final concentration was added and incubated at 37°C for 30 min, followed by inactivation with Tris-HCl pH 7.9, 5 mM EDTA, 50 mg/mL proteinase K at 55°C for 2 hours.  The immunoprecipitated DNA fragments were purified by phenol:chloroform extraction, followed by ethanol precipitation.  The purified ChIPed DNA was processed to prepare ChIP-seq libraries. ChIP-seq libraries were generated from two biological replicates for each condition as described previously (Franco et al., 2015; Luo et al., 2014).  Immunoprecipitated (ChIPed) DNA was purified further by polyethylene glycol (PEG)-mediated precipitation (Lis and Schleif, 1975) using PEG Precipitation Solution (20% in 20 mM Tris-HCl pH 7.9, 4 mM EDTA, 2.5 M NaCl).  After purification, 5 ng (for ERa) and 10 ng (for H3K27ac and H3K4me1) of ChIPed DNA for each treatment condition was used to generate libraries for sequencing, as previously described with some minor modifications (Luo et al., 2014).  Briefly, the ChIPed DNA was end-repaired and a single “A”-base overhang was added using the Klenow fragment of E.coli DNA polymerase.  The A-modified DNA was ligated with Illumina sequencing adapters using the Illumina TruSeq DNA Sample Prep Kit.  The ligated DNA (250 ± 25 bp) was size-selected using a bead-based method with magnetic beads (AmPure XP beads, Beckman Coulter) isolate DNA fragment sizes of interest.  The size-selected DNA was amplified by PCR (9 to 11 cycles as determined empirically) and purified using AmPure XP beads.  The final libraries were run on agarose gel, and extracted using QIAquick gel extraction kit (Qiagen).  The final libraries were subjected to QC (size, purity, adapter contamination).  The quality of libraries was assessed using a High Sensitivity D1000 screen tape on a 2200 TapeStation (Agilent) and quantified using a Qubit dsDNA HS assay kit (ThermoFisher Scientific).  The libraries with unique adapter barcodes were multiplexed and sequenced on an Illumina HiSeq 2500 (single-end, 75 base reads) to a depth of ~40 M reads per condition.