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| Status |
Public on Jan 13, 2017 |
| Title |
input vehicle treated control |
| Sample type |
SRA |
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| Source name |
Normal human primary foreskin fibroblasts (BJ)
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| Organism |
Homo sapiens |
| Characteristics |
treatment: vehicle treated control
cell type: primary foreskin fibroblasts
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| Treatment protocol |
Vitamin D (1,25D 100 nM) resuspended in FBS was added to BJs growing in culture. FBS alone was added to the control sample. 24 h after initiating treatment, cells were crosslinked, sonicated, and chromatin isolated to perform ChIP analysis as described in Gonzalez-Suarez et al. EMBO J 2009. .
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| Growth protocol |
Normal human primary foreskin fibroblasts (BJ) immortalized with telomerase were grown in culture in complete DMEM-HG media, containing 10% FBS, antibiotic, and antimycotics.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
BJ cells were grown to 80% confluence, cross-linked by addition of 36% formaldehyde directly to the cell culture media to a final concentration of 1%, and incubated for 10 minutes at room temperature on a rocking platform. The reaction was stopped by addition of glycine pH 7.0 to a final concentration of 0.125 M. We washed cross-linked cells twice with cold phosphate-buffered saline, scraped them and lysed them at a density of 5X106 cells ml-1 for 10 min at 4°C in 1% SDS, 50 mM Tris-HCl (pH 8.0) and 10 mM EDTA containing protease inhibitors. We sonicated lysates to obtain chromatin fragments <1 kb and centrifuged them for 15 min in a microfuge at room temperature. We diluted lysate 1:10 with 1.1% Triton-X100, 2 mM EDTA, 150 mM NaCl and 20 mM Tris-HCl (pH 8.0) containing protease inhibitors, and precleared the lysates with blocked magnetic beads (Dynabeads with protein-G blocked in 1X PBS+0.5% BSA in dilution buffer). We incubated the fragments with 10 μg of VDR antibody or 10 μg of control IgG, together with 80 μl of Dynabeads mix at 4 °C overnight on a rotating platform. Next day, we washed the immunoprecipitates with 0.1% SDS, 1% Triton-X100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl (one wash); 0.1% SDS, 1% Triton-X100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0) and 500 mM NaCl (one wash); 0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA and 10 mM Tris-HCl, pH 8.0 (one wash); and 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (two washes). We then eluted the chromatin from the beads twice by incubation with 250 μl 1% SDS and 0.1 M NaHCO3 during 15 min at room temperature with rotation. After adding 20 μl of 5 M NaCl, we reversed the crosslinks for 4 h at 65 °C. Samples were supplemented with 20 μl of 1 M Tris-HCl (pH 6.5), 10 μl of 0.5 M EDTA, 20 μg of RNase A and 40 μg of proteinase K and incubated for 1 h at 45 °C. We recovered DNA by phenol-chloroform extraction and ethanol precipitation. For Input samples, we collected 20 μl of chromatin after sonication, and processed these with the rest of the samples at the point of reverse crosslinking.
Ion Torrent IonXpress barcoded sequencing libraries were constructed using the Ion Xpress Plus Fragment Library Kit (Life Technologies) according to the manufacturer's directions except that size selection was performed using Beckman Agencourt magnetic beads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Ion Torrent Proton |
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| Data processing |
Basecalling was performed by Torrent Suite version 4 software using default settings
Aligmentment to the human genome sequence was performed using the TMAP map4 algorithm without soft clipping (-g 3 option)
Aligned bam files for replicate sequencing of each sample were merged, and then separated into individual chromosomes using SAMtools
Genome coverage at each base pair for Input and each ChIP sample was calcluted using BEDtools genomecoveragebed (-d option)
Enrichment in ChIP sample coverage relative to Input coverage in sliding 250 bp windows with 50 bp steps was calculated using custom R scripts as described in Swain et al. submitted. Coverage in each window was normalized to total genome coverage prior to normalization to Input.
Genome_build: hg19
Supplementary_files_format_and_content: sgr text graph files showing ChIP enrichment normalized to input
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| Submission date |
Jan 14, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Susana Gonzalo |
| E-mail(s) |
sgonzalo@slu.edu
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| Phone |
314-997-9244
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| Organization name |
Saint Louis University
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| Department |
Biochemistry and Molecular Biology
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| Street address |
1100 S Grand Blvd
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| City |
St Louis |
| State/province |
MO |
| ZIP/Postal code |
63104 |
| Country |
USA |
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| Platform ID |
GPL17303 |
| Series (2) |
| GSE76884 |
Vitamin D/Vitamin D Receptor (VDR) Axis Regulates LMNA gene expression [ChIP-Seq] |
| GSE76887 |
Vitamin D/Vitamin D Receptor (VDR) Axis Regulates LMNA gene expression |
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| Relations |
| BioSample |
SAMN04414856 |
| SRA |
SRX1531612 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not applicable for this record |
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