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| Status |
Public on Feb 11, 2018 |
| Title |
siHdac3_H3k27acrn5 |
| Sample type |
SRA |
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| Source name |
Schwann cell
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| Organism |
Rattus norvegicus |
| Characteristics |
cell type: Schwann cell
antibody: H3k27ac (Abcam, ab4729)
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| Growth protocol |
primary rat schwann cell progenitors were induced to differentiate for 9 hours in the presence of cAMP
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and proteine-DNA complexes were isolated with antibody.
Schwann cells (~20 million cells) were cross-linked in 1% formaldehyde at room temperature for 10 min and then quenched with 125 mM glycine for 5 min. Cells were rinsed, resuspended in lysis buffer (50 mM HEPES, pH 7.6, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.25% Triton X-100 and 0.5% NP-40 and protease inhibitor cocktail) and incubated on ice for 10 min. Lysates were centrifuged and pellets were resuspended in nuclei washing buffer (200 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM Tris pH 8.0, and protease inhibitor cocktail) with gentle rocking at 4°C. Nuclei were pelleted and resuspended in 1 ml of sonication buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.5 mM EGTA and protease inhibitor cocktail). Nuclear suspensions were sonicated with a Covaris S220 sonicator (total time 8 min). Antibody (chromatin: antibody = 4:1) was added to chromatin and incubated at 4°C overnight. Chromatin-protein complex was immunoprecipitated with protein A/G plus agarose beads and washed sequentially, twice with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.0], 150 mM NaCl), twice with high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.0], 500 mM NaCl) and twice with LiCl buffer (0.25 M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid, 1 mM EDTA, 10 mM Tris [pH 8.0]) and once with 1xTE buffer. Immunoprecipitates were then eluted in elution buffer at 65°C for 1 h. Eluted chromatin was subjected to reverse-crosslink and chromatin fragment purification with QIAGEN PCR purification columns. ChIP-Seq libraries were prepared using NEBNext ChIP-Seq Library Prep Kit according to manufacturer’s instructions.
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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 |
Basecalls performed using CASAVA
ChIP-seq reads were aligned to the rn5 genome assembly using Bowtie2 with the default configuration
Peak calling was performed using MACS (Model-based Analysis of ChIP-Seq) (http://liulab.dfci.harvard.edu/MACS) with a p value cut off of 1x e9.
Genome_build: rn5
Supplementary_files_format_and_content: bed files were generated using Macs14
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| Submission date |
Nov 03, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Richard Lu |
| Organization name |
Cincinnati Children's Hospital Medical Center
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| Department |
CBDI
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| Lab |
Lu Lab,T6.525
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| Street address |
3333 Burnet Ave
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| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
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| Platform ID |
GPL14844 |
| Series (2) |
| GSE93160 |
A Histone Deacetylase 3-Dependent Pathway Delimits Peripheral Myelin Growth and Functional Regeneration [ChIP-Seq] |
| GSE93161 |
A Histone Deacetylase 3-Dependent Pathway Delimits Peripheral Myelin Growth and Functional Regeneration |
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| Relations |
| BioSample |
SAMN07974188 |
| SRA |
SRX3358571 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2838633_siHdac3_H3k27acrn5.wig.gz |
240.5 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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