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| Status |
Public on Feb 20, 2017 |
| Title |
Input_ChIP |
| Sample type |
SRA |
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| Source name |
melan-Ink4a-Arf-null
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| Organism |
Mus musculus |
| Characteristics |
strain: Cdkn2a tm1RDp/tm1RDp mice
cell line: melan-Ink4a-Arf-null
cell type: immortalized melanocyte cell line
chip antibody: none (input)
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| Treatment protocol |
Cells were grown in 1% oxygen for 24hr prior to crosslinking
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| Growth protocol |
According to guidelines from Bennett et al. 1987 (PMID 3102392), cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco), 200 nM 12-O-tetradecanoylphorbol 13-acetate (Sigma-Aldrich, St Louis, MO, USA) and 200 pM cholera toxin (Sigma- Aldrich, St Louis, MO, USA) at 37°C in humidified air.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 2 x 10^7 cells were harvested and crosslinked at room temperature with 1% formaldehyde for 9 minutes. Crosslinking reaction was quenched with the addition of glycine to a final concentration of 125 mM for at least 15 minutes. Crosslinked cells were lysed on ice using 1 ml low-salt ChIP buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.5% NP-40, 1.0%Triton X-100) and sonicated using a Qsonica Sonicator Q500 (Newtown, CT 0647) with the following settings: 70% amplitude; 10 seconds on/20 seconds off cycles; total sonication time of 15 minutes to yield fragments of 500-100 bp. Sonicated samples were centrifuged and the soluble chromatin (supernatant) was used for chromatin immunoprecipiation reactions. Briefly, 200 µl of lysate (~ 2 x 106 cells) was incubated with each antibody, 10µg / IP of HIF1A antibody #AF1935 Immunoprecipitated chromatin was recovered using recombinant protein G beads or magnetic Dynabeads® (Invitrogen). ChIP samples were reverse crosslinked and DNA was purified using the phenol-chloroform extraction method.
Illumina ChIP Seq libraries were prepared as follows: ChIP-isolated DNA was size selected by 2% Agarose Nusieve gel, and a 200-500bp region was excised and purified using Qiaquick gel extraction kit. Adapter linker attachment was performed by PCR using 30ng of DNA and 2X Phusion master mix and primers, using conditions of 30 sec, 98°C, followed by 10 sec, 98°C, 30 sec, 65°C and 30 sec, 72°C with cycle number empirically determined, followed by a final 5 min, 72°C extension step. Adapter ligated PCR reactions were purified using Agencourt Ampure XP PCR Purification Beads per manufacturer’s protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
ChIP-Seq peaks for each experimental replicate were called independently using the Model-based Analysis for ChIP-Seq (MACS v1.4.2) algorithm by applying default settings and a significance p-value threshold of 1.00e-05 and >10-fold enrichment over background.
Only ChIP-Seq profiles that showed reproducible overlap between both experimental samples were used for downstream analyses.
Genome_build: mm9
Supplementary_files_format_and_content: Wig files were generated using MACS and scores represent tag pileup over each position. Text files represent narrow peak file ouput from MACS
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| Submission date |
Sep 07, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Stacie Loftus |
| E-mail(s) |
sloftus@mail.nih.gov
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| Phone |
301-594-1752
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| Organization name |
National Human Genome Research Institute
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| Street address |
49 Convent Dr
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| City |
Bethesda |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE86554 |
Identification of hypoxia-induced HIF1A targets in melanocytes reveals a molecular profile associated with poor prognosis for melanoma [ChIP-seq] |
| GSE86555 |
Identification of hypoxia-induced HIF1A targets in melanocytes reveals a molecular profile associated with poor prognosis for melanoma |
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| Relations |
| BioSample |
SAMN05750469 |
| SRA |
SRX2146747 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2305572_Input_ChIP_processed.wig.gz |
382.3 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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