title: GSM1027487: POB_H3K4me3125; Mus musculus; ChIP-Seq
library_construction_protocol: Chromatin immunoprecipitation was performed as described previously (Meyer MB. Mol Endo. 2012).  Briefly, MC3T3-E1 cells were treated for 3 hrs with vehicle or 100nM 1,25(OH)2D3.  Samples were subjected to immunoprecipitation using either a control IgG antibody or the indicated experimental antibody (VDR, RXR, C/EBPβ, RUNX2, H3K5Ac, H3K9Ac, H3K4me1, H3K4me3, or H3K36me3).  For differentiated cells, there was substantial mineralized matrix.  To remove the matrix from the assay, the cell/matrix mix was subjected to 3x 15s pulses with a Polytron Homogenizer (Power Gen 125, Fisher Scientific) while in NCP 1 buffer (Hepes 10mM pH 6.5, EDTA 10mM, EGTA 0.5mM, Triton X-100 0.25%).  The resulting homogenate was centrifuged (200 x g) through a 10% sucrose buffer at a 1:1 ratio (10mM Tris-HCl, 15mM NaCl, 60mM KCl, 1mM EDTA, 10% Sucrose).  Resulting cell pellet was resuspended in NCP 2 buffer (Hepes 10mM pH 6.5, EDTA 1mM, EGTA 0.5mM, NaCl 200mM) and remainder of protocol was followed (Meyer MB. Mol Endo. 2012).  The isolated DNA (or Input DNA acquired prior to precipitation) was then validated by quantitative real time PCR (qPCR) and further prepared for ChIP-seq analysis.  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: GSM1027487