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| Status |
Public on Jul 14, 2016 |
| Title |
TKS184 E14 H3K27me3 coverage |
| Sample type |
SRA |
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| Source name |
wild-type E14 cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic cell line E14
genotype/variation: wild type
passage/age: passage 3 post-transfection
chip antibody: H3K27me3 (Millipore, 07-449)
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| Growth protocol |
E14 cells were maintained in ES medium (knock-out DMEM, 20% fetal bovine serum, 55 µM mercaptoethanol, 1 mM sodium pyruvate, 2 mM glutamine, 0.1 mM nonessential amino acids, and 1000 U/ml mouse LIF), on tissue culture plates coated with 0.1% gelatin, in 6% CO2 at 37°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells for ChIP were crosslinked in 1% formaldehyde for 15 min at room temperature, followed by quenching of the formaldehyde in 0.125 M glycine for 5 min. After washing the cells with PBS, they were scraped in cell lysis buffer (4% SDS, 50 mM Tris-HCl at pH 8.0, 10 mM EDTA, 100 mM NaCl, 1 mM PMSF) containing protease inhibitor (Roche). The cell lysate was incubated for 30 min at room temperature, and the crude extract was loaded on top of 8 M urea followed by ultracentrifugation overnight. The pure chromatin pellets were collected in dialysis buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.5 mM EGTA, 5% glycerol 0.1% Triton X-100), and dialyzed for 16 hrs at 4°C. Chromatin recovered from dialysis was fragmented to 300–800 bp by restriction enzyme digestion followed by sonication in a Bioruptor (Diagenode, high power, 30 sec on and 30 sec off for 15 cycles) in sonication buffer (0.1% SDS and 0.1% DOC in dialysis buffer). To separate insoluble components, we centrifuged the samples at 13,000 rpm for 5 min at 4°C and recovered the supernatant and pellet separately. The pellets were dissolved in sonication buffer, sonicated again for 15 cycles in a Bioruptor on high power, and centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was combined with the previous one, and a reverse-crosslinked aliquot was examined for chromatin shearing efficiency on a Bioanalyzer 2100 (Agilent Technologies Inc.). Reverse crosslinking of chromatin was done at 55°C overnight in buffer containing 1% SDS, 200 mM NaCl, 10 mM EDTA and 200 µg/ml proteinase K followed by incubation with 100 µg/ml RNase A at 37°C for 1 hr. DNA was recovered by using QIAquick PCR purification kit (Qiagen).
DNA from ChIP and input chromatin fractions were sheared to an average size of approximately 300 bp using a Bioruptor UCD-200 (Diagenode) and verified on a Bioanalyzer 2100 (Agilent). For each ChIP-seq experiment, we used a custom library construction protocol. Briefly per library, ChIP DNA fragments were end-repaired and ligated to indexed sequencing adapters and amplified with 15 rounds of PCR. The amplified library was selected for fragments over 200 bp in length using Agencourt AMPureXP or SPRI beads.
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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 |
Raw ChIP-seq data was aligned to mouse genome release mm9 using bowtie v1.0.0 with the following parameters set: --chunkmbs 1024 --best --strata -n 3 -m 1
Candidate peaks were called using MACS (v1.4.3) using default settings.
Genome_build: NCBI37/mm9
Supplementary_files_format_and_content: bigWig tracks of coverage profiles were generated using the wigToBigWig utility from UCSC. The bigWig track of MACS peaks was created using a custom Perl script.
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| Submission date |
Jul 18, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Hunter Richards |
| E-mail(s) |
hrichards@ucsf.edu
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| Organization name |
UCSF
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| Department |
Orofacial Sciences
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| Lab |
Kohwi-Shigematsu
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| Street address |
513 Parnassus Ave., HSW 863
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94103 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE70974 |
Tip5/Baz2a Regulates Chromatin Architecture and Gene Expression to Maintain Self-renewal and Pluripotency of Embryonic Stem Cells [ChIP-seq] |
| GSE70975 |
Tip5/Baz2a Regulates Chromatin Architecture and Gene Expression to Maintain Self-renewal and Pluripotency of Embryonic Stem Cells. |
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| Relations |
| BioSample |
SAMN03879902 |
| SRA |
SRX1100370 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1826465_TKS184_E14_H3K27me3_coverage.bw |
513.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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