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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 10, 2015 |
| Title |
HCT116-WT-IgG |
| Sample type |
SRA |
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| Source name |
HCT116 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116 colon cancer cells
genotype/variation: WT
medip antibody: IgG (Rat)(Diagenode, catalog# AF-110-0016, lot# 004)
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| Growth protocol |
HCT116-WT and HCT116-DKO (DKO1 line) cells were generous gifts from Dr. B. Vogelstein (Johns Hopkins University). These cells were cultured in McCoy's 5A medium with 10% FBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA (1 μg) was isolated from cultured cells and postmortem brain tissue using the AllPrep DNA/RNA kit (Qiagen) according to the manufacturer’s recommendations for cells and tissues. DNA concentration was determined using the Qubit DNA Broad Range fluorescence assay (Life Technologies) and Qubit Instrument (Life Technologies).
MeDIP-Seq libraries for Ion Torrent semiconductor sequencing on the Ion Torrent PGM or Ion Torrent Proton instruments were generated using a modified protocol consisting of Life Technologies’ Ion Plus Fragment Library kit (Catalog # 4471252) in combination with a commercially available 5-methylcytosine (MagMeDIP Kit, Catalog # mc-magme- 048) or 5-hydroxymethylcytosine (5hmC Kit, Catalog # AF-110-0016) immunoprecipitation kit (Diagenode). A nonspecific mouse IgG was used for an immunoprecipitation reaction as a negative control. For both 5-mC and 5-hmC MeDIPSeq, 1 ug of DNA was sheared to a mean fragment size of 300 bp by sonication with the Covaris M220 Focused-ultrasonicator instrument (Covaris) in a 50 μl microTUBE AFA Fiber Screw-CAP (Covaris). Sonicated DNA was end-repaired, purified with Agencourt AMPure XP reagent beads (Beckman Coulter), and ligated with Ion Torrent compatible sequencing adapters following the standard Ion Plus Fragment Library Kit protocol. Adapter-ligated DNA was purified using Agencourt AMPure XP reagent beads and immunoprecipitated using either a 5-methylcytosine kit (MagMeDIP) or 5- hydroxymethylcytosine (hMeDIP) MeDIP kit according to the manufacturer’s protocol. Following either 5-mC or 5-hmC MeDIP, libraries were size-selected from 200-400 bp on a 2% agarose E-gel (Invitrogen) and extracted using a MinElute Gel DNA extraction kit (Qiagen) according to the manufacturer’s instructions. Libraries were then amplified at 18 cycles of PCR (95°C, 15 s; 58°C, 15 s; 70°C, 1 min), purified using MinElute columns (Qiagen), and eluted in 16 μl EB (Qiagen). Libraries were assessed for quality and concentration on an Agilent Bioanalyzer instrument. An aliquot of each library was used to assess enrichment efficiency for control methylated DNA and hydroxymethylated DNA compared to 10% input DNA.
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| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Ion Torrent Proton |
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| Data processing |
Torrent Suite v4.0 used to perform base calling.
Reads were trimmed for adapter sequence, poly-clonal, and low quality then aligned to the hg19 assembly using TMAP.
BAM files of mapped MeDIP-seq reads and IgG reads were used with MACS v2.
Genome_build: hg19
Supplementary_files_format_and_content: bed files of MACS peaks called
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| Submission date |
Oct 01, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Michael Corley |
| E-mail(s) |
mjc4002@med.cornell.edu
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| Organization name |
Weill Cornell Medicine
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| Department |
Medicine, Division of Infectious Diseases
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| Street address |
413 East 69th Street
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10021 |
| Country |
USA |
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| Platform ID |
GPL17303 |
| Series (1) |
| GSE61968 |
Semiconductor-based sequencing of genome-wide DNA methylation states |
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| Relations |
| BioSample |
SAMN03086658 |
| SRA |
SRX719222 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
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