title: GSM1263823: Liver_ERa_INPUT_E2_repl1; Mus musculus; ChIP-Seq
library_construction_protocol: Samples were cross-linked with 1% formaldehyde at 37°C for 15 minutes and quenched with 0.125M glycine.  Tissues were then rinsed with 1X PBS, minced with scissors, and homogenized.  Samples were pelleted by brief centrifugation and resuspended in 0.5 ml lysis buffer (1% SDS, 10mM EDTA pH 8.0, 50mM Tris pH 8.0, 1x Complete Protease Inhibitors (Roche 1187358001)).  DNA was fragmented by seven rounds of sonication with a Sonifier 250 tip sonicator (Branson Ultransonics) at output setting 5 for 5 seconds followed by 1 minute on ice.  Samples were centrifuged at 13500 rpm for 15 minutes at 4°C and the supernatant collected.  14.5 l of Protein A Dynabeads and 14.5 l of protein G Dynabeads (invitrogen) were washed by magnetic pull down 3x at 4oC with PBS+5mg/ml BSA. 15 l of anti-ER HC-20 antibody (Santa Cruz) and 10 l of anti-ER Ab-10 (Neomarkers) were incubated with the beads for 6 to 8 hours. Beads were washed 2x w/ PBS+BSA to remove unbound antibody, and resuspended in 0.5ml PBS+BSA. 280l of fixed chromatin (in sonication buffer) was mixed with 1.12 ml of Dilution Buffer (1% Triton X-100, 2mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8.1) and added to the beads, followed by overnight incubation with rocking at 4oC. Beads were collected by magnetic pull down and washed 6X with RIPA buffer (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 0.7% Sodium Deoxycholate, 1% NP40, 0.5M LiCl) using 20 minute and 5 minute alternating wash times, and twice with TE for 5 minutes. DNA was released and cross-links reversed by resuspending in Elution Buffer (100mM NaHCO3, 1% SDS) and incubating at 65oC overnight (vortexing every 5 minutes for the first 30 minutes). Samples of input chromatin were also resuspended in elution buffer and incubated at 65oC to reverse crosslinks. DNA was purified using the Qiaquick PCR kit (Qiagen) and quantified using the Quant-IT dsDNA HS kit (Invitrogen). 4ng of input and ChIP samples from two biological replicates were prepared for sequencing essentially using the standard Illumina protocol, with the following modifications: we used the End-It kit (Epicentre Biotech.) for end repair, Taq polymerase for A addition, and Easy A thermostable polymerase (Agilent Technologies) for the final limited amplification. Ligated or final amplified products of between ~220 and ~320 bp size were isolated by E-gel or 1.6% agarose gel electrophoresis followed by Qiagen Gel Extraction kit purification. For all other steps, products were purified by addition of 30 mg glycogen (invitrogen #1286677) and EtOH precipitation, followed by a 70% EtOH wash. The library was quantified using the Quant-IT dsDNA HS kit (Invitrogen) and approximately 10 ng submitted for sequencing. First replicate samples were not multiplexed. 2nd replicate samples were multiplexed using Illumina multiplex adapters and run together on one HiSeq lane (Aorta_IP=Barcode 2, Aorta INPUT=barcode 6, Liver IP=Barcode 4, Liver input= barcode 8). Raw data files were de-multiplexed by the sequencing facility.
attributes.tag: GEO Accession
attributes.value: GSM1263823