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| Status |
Public on Nov 16, 2012 |
| Title |
Oct4 ChIP-Seq at 48hrs Post-Induction |
| Sample type |
SRA |
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| Source name |
BJ_Oct4 ChIP-Seq_48hr_postinduction
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| Organism |
Homo sapiens |
| Characteristics |
cell line: BJ
cell type: foreskin fibroblast cell
genotype/variation: infected with lentiviruses encoding for dox-inducible Oct4, Sox2, Klf4, and c-Myc, along with lentiviruses expressing rtTA2M2
time point: 48hrs post-induction with with 1µg/ml dox
chip antibody: Oct4
chip antibody vendor: abcam
chip antibody cat. #: ab19857
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| Treatment protocol |
Lentiviral production and titration is described in the Supplemental Experimental Procedures. BJ cells at passage 10 were infected with lentiviruses encoding for dox-inducible Oct4, Sox2, Klf4, and c-Myc, along with lentiviruses expressing rtTA2M2 in the presence of 4.5 µg/ml polybrene. The expression of the OSKM factors was induced by treating the infected BJ cells with 1µg/ml dox for 48 hours. Cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature. BJ cells (MOCK, not-infected) were treated with 1µg/ml dox for 48 hours.
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| Growth protocol |
The human foreskin fibroblast cell line (BJ) were obtained from the American Type Culture Collection (ATCC # CRL-2522) at passage 6 and cultured in the ATCC-formulated Eagle's Minimum Essential supplemented with 10% FBS at 37C and CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was obtained from BJ cells infected with OSKM plus rtTA2M2 lentiviruses and induced with dox for 48hrs. Chromatin fragments associated with Oct4, Sox2, Klf4, or c-Myc were prepared from eight pooled replicate populations of 10e7 OSKM-infected cells, as described previously in (Boyer et al., 2005), using 10 µg of specific antibodies raised against Oct4 (abcam # ab19857), Sox2 (R&D # AF2018), Klf4 (R&D # AF3640), and c-Myc (R&D # AF3696). DNA libraries were constructed from ~ 20 ng of DNA obtained from each ChIP of Oct4, Sox2, Klf4 and c-Myc expressed for 48 hr in OSKM-infected fibroblasts and from non-immunoenriched genomic DNA (input DNA) of OSKM infected fibroblasts using the Illumina library kit (illumina # 11257047). The DNA libraries were assessed using on Agilent Technologies 2100 Bioanalyzer and found to be in the 150-300 bp size range.
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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 |
DNA immunoprecipitated by Oct4 (abcam # ab19857)
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| Data processing |
Base-calling: Illumina software version 1.70
Alignment: ELAND, default parameters
Filtering: non-unique aligned sequence tags were discarded down to a single tag
Peak-calling: MACS, MFOLD = 16 using input as background
Filtering: peak sets were filtered to control the FDR at 0.5%
Multiple Transcription Factor Binding Regions (MTFBRs) were called using the algorithm described in the methods
Enrichment: bigWig files for each O, S, K, and M mark were created in the following way. Aligned, unique tags were binned in 25 bp intervals. Each interval count was normalized by RPKM (a measurement of the number of billions of bases sequenced per lane) and a normalized input count for the same interval was subtracted. The resulting interval enrichment scores were written to a bedGraph file, which was turned into a bigWig using the bedGraphToBigWig program from UCSC Genome Browser's online utilities package.
Genome_build: hg18
Supplementary_files_format_and_content: Tag-Aligned Files: BED6 files containing six tab-delimited columns: chromosome, start, stop, tag ID, null, strand (post-filtering for non-unique reads)
Supplementary_files_format_and_content: Peak Files: BED6 files containing six tab-delimited columns: chromosome, start, stop, null, null, strand (contains final, FDR-filtered peak sets only)
Supplementary_files_format_and_content: BigWig Files: 25 bp binned aligned, unique tags (binary data), per-lane normalized and input subtracted
Supplementary_files_format_and_content: DBRs: BED6 files containing the loci of the OSKM differentially-bound regions between 48 hrs reprogrammed fibroblasts and ES cells. A Series-specific BED file containing large, megabase-scale loci where no TFs bind.
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| Submission date |
Mar 16, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Kenneth S. Zaret |
| E-mail(s) |
zaret@pennmedicine.upenn.edu
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| Phone |
2155735813
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| Organization name |
University of Pennsylvania School of Medicine
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| Department |
Cell and Developmental Biology
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| Lab |
Zaret lab
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| Street address |
9-132, SCTR, 3400 Civic Center Boulevard
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104-5157 |
| Country |
USA |
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| Platform ID |
GPL10999 |
| Series (1) |
| GSE36570 |
OSKM factors cooperatively engage chromatin to initiate reprogramming |
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| Relations |
| SRA |
SRX130060 |
| BioSample |
SAMN00828870 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM896985_Oct4_48hrs.bed.gz |
289.0 Mb |
(ftp)(http) |
BED |
| GSM896985_Oct4_48hrs.bw |
302.0 Mb |
(ftp)(http) |
BW |
| GSM896985_Oct4_48hrs_peaks.bed.gz |
476.0 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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