title: GSM1694862: Intestine_1,25_3; Mus musculus; ChIP-Seq
library_construction_protocol: Intestinal epithelial cells were isolated using EDTA as reported previously (Sato, T. et al, (2009) Nature 459:262). In brief, collected proximal half site of small intestines were cleaned with cold PBS and sliced into small pieces. The tissues were then incubated in cold PBS containing 2 mM EDTA on ice for 30 minutes and washed with cold PBS. Epithelial cells were isolated by vortexing the tissues with cold PBS 5 times for 1.5 minutes with 30-seconds intervals on ice and then by passing the mixture through 70-micrometer cell strainer. The isolated cells were centrifuged at 2000 rpm for 5 minutes, washed with cold PBS and subjected to chromatin immunoprecipitation using either a control IgG antibody or anti-VDR antibody as described previously (Meyer, M. B. et al (2012) Mol Endocrinol 26:37). 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 GAIIx [for all samples sequenced before June, 2011] or the Illumina HiSeq2000 [after June, 2011] 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) 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) 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.  Each ChIP sample was repeated in biological replicate in the same manner (minimum of 2 replicates).  Sequences were mapped to the mouse genome (mm9) using BOWTIE (--best –m 1) to yield unique alignments.
attributes.tag: GEO Accession
attributes.value: GSM1694862