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| Status |
Public on Jun 03, 2015 |
| Title |
Intestine_1,25_2 |
| Sample type |
SRA |
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| Source name |
proxmial half site of small intestine
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| Organism |
Mus musculus |
| Characteristics |
tissue: duodenum and jejunum
treatment: 1,25(OH)2D3 (10 ng/g bw)
antibody: anti-VDR (Santa Cruz Biotechnology; sc-1008)
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| Treatment protocol |
Mice at 8 weeks of age were treated with vehicle or 10 ng/g body weight of 1,25(OH)2D3 for 1 hours prior to tissue harvesting.
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| Growth protocol |
Wildtype mice were fed a standard rodent chow diet (Harlan Teklad, #5008).
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| Extracted molecule |
genomic DNA |
| Extraction 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.
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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: *.bed files contain the ChIP-seq peak locations, *.bedgraph files are for display in the UCSC genome browser
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| Submission date |
May 22, 2015 |
| 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) |
| GSE69179 |
1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium Regulating Components (ChIP-seq) |
| GSE69180 |
1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium Regulating Components |
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| Relations |
| BioSample |
SAMN03732369 |
| SRA |
SRX1037021 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1694861_Intestine_VDR_125_rep2.peaks.bed.gz |
383.7 Kb |
(ftp)(http) |
BED |
| GSM1694861_Intestine_VDR_125_rep2.tagdirnew.ucsc.bedGraph.gz |
31.4 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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