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| Status |
Public on May 24, 2013 |
| Title |
CTCF_NPC_ChIPSeq |
| Sample type |
SRA |
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| Source name |
Neural progenitor cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: ES-derived neural progenitor cells
strain: 129SvJae x C57BL/6
gender: male
chip antibody: CTCF (Millipore; 07-729 DAM1772428)
catalog/lot#: 07-729 DAM1772428
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| Treatment protocol |
V6.5 ES cells were differentiated into neural progenitor cells (NPCs) using established techniques (Meissner et al., 2008; Mikkelsen et al., 2007; Okabe et al., 1996). Briefly, after expansion, ES cells were trypsinized and cultured in bacterial dishes for 4 days in the absence of LIF to promote embryoid body formation. Embryoid bodies were then plated on tissue-culture plastic dishes and cultured in serum-free, defined media (ITSFn). After 5-7 days selection in ITSFn, adherent cells were trypsinized, triturated to a single cell suspension, and re-plated on tissue culture dishes coated with 15 ug/ml poly-L-ornithine (Sigma) and 1 ug/ml human plasma fibronectin (Gibco). NPCs were further propagated for 2-4 days in DMEM/F12 supplemented with 25 ug/ml insulin, 50 ug/ml human APO transferrin, 30 nM sodium selenite, 20 nM progesterone (Sigma), 100 nM putrescine (Sigma), 1 ug/ml laminin (Sigma), 10 ng/ml Fgf2 (R&D Systems), and 1x penicillin/streptomycin. NPCs were ~60-75% confluent at the time of fixation with 1% formaldehyde before downstream assay.
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| Growth protocol |
Murine V6.5 ES cells were expanded on Mitomycin-C-inactivated MEF feeder layers in pluripotent media supplemented with LIF. After initial expansion, ES cells were passaged 2-3 times on 0.1% gelatin to remove contaminating feeder cells.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation (ChIP) was performed as previously described with minor modifications (Kagey et al., 2010). Briefly, 50x106 NPCs were lysed and chromatin was sonicated with a Branson Sonifier 250 for 30 x 12 sec pulses. Chromatin was pre-cleared with Protein A Sepharose beads and then immunoprecipitated with approximately 10 μg of the appropriate antibody coupled to Protein A sepharose beads. For Smc1a ChIP using Bethyl Laboratories (A300-055A) affinity purified rabbit polyclonal antibody, beads were washed 1X with LB3, 1X with 20mM Tris-HCl pH8, 500mM NaCl, 2mM EDTA, 0.1% SDS, 1%Triton X-100, 1X with 10mM Tris-HCl pH8, 250nM LiCl, 2mM EDTA, 1% NP40 and 1X with TE containing 50 mM NaCl. For CTCF ChIP using an Upstate 07-729 rabbit polyclonal antibody, beads were washed 7 times with RIPA buffer and 1X with TE containing 50 mM NaCl. Bound complexes were eluted from the beads by heating at 65°C for 15 min with occasional vortexing. Cross-links were reversed by overnight incubation at 65°C. Whole cell extract DNA reserved from the sonication step was also treated for crosslink reversal. ChIP-seq libraries were prepared for sequencing as recommended by Illumina/Solexa.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
ChIP-seq
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| Data processing |
Sequences were aligned to NCBI Build 37 (UCSC mm9) using default parameters (-v1 -m1) in Bowtie. Only sequences that mapped uniquely to the genome were used for further analysis. Model-based Analysis for ChIP-Sequencing (MACS) was used for peak calling (http://liulab.dfci.harvard.edu/MACS/00README.html) (Zhang et al., 2008). Default parameters were used with a p-value cutoff of P<1E-8. A background whole cell extract input control was used to assess significance.
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| Submission date |
Mar 01, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Jennifer Elizabeth Phillips-Cremins |
| E-mail(s) |
Jennifer.Phillips-Cremins@umassmed.edu
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| Organization name |
University of Massachusetts Medical School and Emory University
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| Department |
Program in Systems Biology and Department of Biology
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| Lab |
Job Dekker Laboratory and Victor Corces Laboratory
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| Street address |
364 Plantation Street
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| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE36203 |
Architectural protein subclasses shape 3-D organization of genomes during lineage commitment |
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| Relations |
| SRA |
SRX125274 |
| BioSample |
SAMN00794613 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM883647_Phillips_NPC_CTCF_pe1_pvalue1E-8_mm9_peaks.bed.gz |
614.8 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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