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| Status |
Public on Sep 05, 2012 |
| Title |
LS180_MED1_Veh |
| Sample type |
SRA |
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| Source name |
LS180_MED1_Veh
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| Organism |
Homo sapiens |
| Characteristics |
cell line: LS180 cells
passage number: 12-23
cell type: cultured CRC cells
antibody: MED1 (Santa Cruz, M-255, sc-8998)
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| Treatment protocol |
Cells were treated with 10-7M 1,25(OH)2D3 [125] or with ethanol vehicle [Veh] for 3 hours prior to ChIP assay
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| Growth protocol |
Cells were grown in normal MEM-alpha with 10% standard FBS serum, 1% Non-essential Amino Acids, 1% Sodium Pyruvate and 1% Penicillin-Streptomycin
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation was performed by treating LS180 cells for 3 hrs with vehicle or 10-7M 1,25(OH)2D3 as was optimized as previously reported. ChIP samples were created from 100 million cells per sample. The treated cells were washed several times with PBS and then subjected to a 15 minute cross-linking reaction with 1.5% formaldehyde. After the cross-linking reaction, isolated cell extracts were sonicated to prepare chromatin fragments (average DNA size of 500-600 bp DNA as assessed by agarose gel electrophoresis) using a Fisher Model 100 Sonic Dismembranator at a power setting of 2. Pre-cleared samples were subjected to immunoprecipitation using either a control IgG antibody or the indicated experimental antibody. The precipitated DNA was then washed, the cross-links reversed and the DNA fragments purified using Qiagen QIAquick PCR Purification Kits (Valencia, CA). The isolated DNA (or DNA acquired prior to precipitation (input)) was then subjected to quantitative real time PCR (qPCR) and further preparation for ChIP-chip or ChIP-seq analysis. ChIP-DNA was prepared and amplified using the Illumina ChIP-seq DNA preparation kit (1003473, #11257047 RevA), clusters formed and sequenced on the Illumina GAIIx sequencers by the University of Wisconsin - Madison DNA Sequencing Facility in the University of Wisconsin- Madison Biotechnology Center. DNA clusters were generated using either a Standard Cluster Generation kit (ver. 4) on an Illumina cluster station (Illumina, San Diego, CA), [for all samples sequenced before April, 2010] or using a cBot Single Read Cluster Generation kit on an Illumina cBot (Illumina) [after April, 2010] according to the manufacturer's instructions, to obtain an average of 2.0×107 clusters for each lane on a flowcell. All sequencing runs for 36mers were performed on an Illumina Genome Analyzer IIx using the Illumina Sequencing kit (ver. 4).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
ChIP-DNA
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| Data processing |
ChIP-seq runs were all 36bp. All samples were mapped from fastq files using Bowtie [-m 1 -- best] to hg18 [UCSC human genome build 18]. Replicate lanes were validated separately for reproducibility and then combined for greater read depth.
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 with the following exception: clonal filter = 5.
False Discovery Rate (FDR) cut off was 0.001 (0.1%) for all peaks.
Genome_build: hg18
Supplementary_files_format_and_content: Bowtie files are the bowtie aligned files, bedGraph files are for display in the UCSC genome browser and the peaks.bed files are the resulting peaks found with QuEST and HOMER
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| Submission date |
Jul 11, 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 |
GPL10999 |
| Series (1) |
| GSE39277 |
Corepressors (NCoR and SMRT) as well as Coactivators are Recruited to Positively Regulated 1α,25-Dihydroxyvitamin D3-Responsive Genes |
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| Relations |
| SRA |
SRX159190 |
| BioSample |
SAMN01087382 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM959629_LS180_MED1_Veh.bedGraph.gz |
49.4 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM959629_LS180_MED1_Veh.bowtie.txt.gz |
841.1 Mb |
(ftp)(http) |
TXT |
| GSM959629_LS180_MED1_Veh_peaks.bed.gz |
12.2 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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