alias: GSM3303982
title: GSM3303982: MCF7_Alarid_2pER_E2_rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: Post-treatment and fixation, nuclei were isolated from MCF-7 cells and sonicated to shear DNA. Protein-DNA complexes were then immunoprecipitated with antibodies specific to either pan-ER or pS118-ER followed by purification of DNA. ChIP-seq libraries were prepared using the NEBNext DNA sample prep kit (NEB, #E6000L) with the Bioo NEXTflex ChIP-seq Barcodes (Bioo Scientific, Austin, TX, #514122) according to manufacturer's protocols, with few exceptions.  During the NEBNext prep, the Illumina adapters were replaced with the Bioo Scientific Barcoded adapters according to Bioo protocols.  ChIP-DNA ligated libraries were cleaned up with Agencourt AMPureXP Magnetic Beads (Beckman-Coulter, #A63881).  A pre-size selection PCR was performed using Phusion polymerase, NEXTflex Primer Mix and purified ligation product for 4 cycles of PCR according to the Bioo Protocol.  Libraries were size selected using Invitrogen E-gels to a size of 400-500bp.  Samples were then PCR amplified for 14 cycles using Phusion polymerase, NEXTflex Primer mix and the size selected DNA as per Bioo protocol, followed by Agencourt bead clean up.  Libraries were validated for integrity using the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).  Clusters were formed and sequenced on the Illumina HiSeq2000 sequencers by the University of Wisconsin-Madison DNA Sequencing Facility in the University of Wisconsin-Madison Biotechnology Center.  DNA clusters were generated using a cBot Single Read Cluster Generation kit (ver.  3) on an Illumina cBot (Illumina, Carlsbad, CA) according to the manufacturer's instructions, to obtain an average of 1.5×108 clusters for each lane on a flowcell.  All sequencing runs for 50mers were performed on an Illumina HiSeq2000 using the Illumina Sequencing kit (ver.  3). Fluorescent images were analyzed using the CASAVA 1.8.2 (Illumina Carlsbad, CA) to obtain FASTQ formatted sequence data.  Twelve barcoded libraries were run per lane and this was repeated over 4 lanes.  After which, the raw FASTQ for each sample was concatenated from the 4 lanes prior to mapping to create a single sample.  The ChIP samples were repeated in biological replicate in the same manner (3 replicates). Sequences were mapped to the human genome (hg19) using BOWTIE (--best –m 1) to yield unique alignments.
identifiers.namespace: GEO
identifiers.id: GSM3303982