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| Status |
Public on Jun 02, 2014 |
| Title |
OB_CEBPb125 |
| Sample type |
SRA |
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| Source name |
MC3T3-E1 cells differentiated for 15 days
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| Organism |
Mus musculus |
| Characteristics |
passage number: 5 - 12
antibody: C/EBPβ (Santa Cruz, C-19, sc-150, lot# F3011)
cell line: MC3T3-E1 differentiated for 15 days
treatment: 100nM 1,25(OH)2D3
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| Treatment protocol |
Cells were treated with 100nM 1,25(OH)2D3 [125] or with ethanol vehicle [Veh] for 3 hours prior to ChIP assay
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| Growth protocol |
MC3T3-E1 cells were cultured in alpha MEM with 10% heat-inactivated fetal bovine serum from Hyclone (Logan, UT) and 1% penicillin-streptomycin. Differentiated cells were cultured in complete media plus 10mM glycerol-2-phosphate and 50ug/mL ascorbic acid for 15 days, changing media every 2-3 days.
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| Extracted molecule |
genomic DNA |
| Extraction 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.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
ChIP-seq runs were all 50bp. Barcoded samples were run over 4 lanes total and the fastq data were concatenated prior to BOWTIE mapping. 12 barcoded samples were run in each lane, over 4 lanes total. Barcodes were decoded by Illumina HiSeq2000 software automatically. All samples were mapped from fastq files using BOWTIE [-m 1 -- best] to mm9 [UCSCmouse genome build 9]. Replicate lanes were analyzed separately for reproducibility and normalization of the peak calls.
Peaks were called by using HOMER [http://biowhat.ucsd.edu/homer/] and QuEST [http://mendel.stanford.edu/sidowlab/downloads/quest/].
QuEST 2.4 was run using the recommend settings for transcription factor (TF) like binding with the following exceptions: kde_bandwith=30, region_size=600, ChIP threshold=30, enrichment fold=3, rescue fold=3
HOMER analysis was run using the default settings for peak finding. Histone peaks were called with a 2-fold enrichment over input instead of 4-fold (for transcription factors) given the nature of histone chip-seq.
False Discovery Rate (FDR) cut off was 0.001 (0.1%) for all peaks.
Genome_build: mm9
Supplementary_files_format_and_content: fastq files are the raw sequence files [barcoded lanes (4 each) were concatenated for each sample for simplicity, no other modification was made], bedGraph files are for display in the UCSC genome browser, *replicate_overlap.bed are the resulting peaks found with QuEST and HOMER after normalization between both replicates
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| Submission date |
Oct 30, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Mark B Meyer |
| E-mail(s) |
markmeyer@wisc.edu
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| Phone |
608-890-0857
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| Organization name |
University of Wisconsin-Madison
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| Department |
Nutritional Sciences
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| Lab |
Meyer Lab
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| Street address |
1415 Linden Dr.
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| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE51513 |
Genomic Determinants of Gene Regulation by 1,25-Dihydroxyvitamin D3 During Osteoblast-Lineage Cell Differentiation [ChIP-Seq] |
| GSE51515 |
Genomic Determinants of Gene Regulation by 1,25-Dihydroxyvitamin D3 During Osteoblast-Lineage Cell Differentiation |
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| Relations |
| BioSample |
SAMN01797411 |
| SRA |
SRX423964 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1027495_MC3T3BONED15_CEBPb125_rep1.tagdirnew.ucsc.bedGraph.gz |
74.3 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM1027495_MC3T3BONED15_CEBPb125_rep2.tagdirnew.ucsc.bedGraph.gz |
67.7 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM1027495_MC3T3BONED15_CEBPb125_replicate_overlaps.bed.gz |
252.5 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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