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| Status |
Public on Nov 15, 2016 |
| Title |
Dnmt1KD_hMeDIP_rep1 |
| Sample type |
SRA |
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| Source name |
murine embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14
antibody: 5-hydroxymethylcytosine
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| Treatment protocol |
One 10cm dish was transiently transfected with esiRNAs and lipofectamine 2000 and cultured 48 hours post transfection.
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| Growth protocol |
E14 mouse embryonic stem cells (ESCs) were cultured on gelatin-coated dishes without feeder cells in high-glucose DMEM medium (Sigma) containing 10% serum (Corning) and LIF at 37°C with 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from ES cells by resuspending cells in ES cell lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM EDTA, 10 mM NaCL, 0.5% sarkosyl), treating with RNase A, and incubating with 1 μg/μL proteinase K overnight at 55°C. DNA was cleaned through PCI extraction and isopropanol precipitation. Genomic DNA was then diluted to 20 ug/mL in TE buffer and sonicated in a Bioruptor (UCD-200) on low for 20 minutes with intervals of 15 seconds on/15 seconds off at 4°C. Sheared DNA (ranging in size from 200-600 bp) was prepared for deep sequencing and further processing by end-repaired, A-tailed, and adaptor-ligated with DNA purification through PCI extraction and ethanol precipitation between each step. 1.5 μg of prepared DNA was diluted in TE buffer up to 450 μL, denatured for 10 minutes at 95°C and immediately transferred to ice for 10 minutes. Denatured DNA was then incubated with 50 μL 10X DNA IP buffer (100 mM Na-HPO4 pH 7.0, 1.4 M NaCl, 0.5% Triton X-100) and 1.5 μL anti-5hmC (Active Motif 39791) overnight at 4°C with constant rotation. 40 μL of magnetic beads (anti-rabbit IgG for 5hmC, Life Technologies) were pre-blocked with BSA and then resuspended in DNA IP buffer. Beads were then added to the DNA/antibody mixture and incubated for 2 hours at 4°C with constant rotation. Beads were then washed three times with 700 μL 1X DNA IP buffer and DNA was eluted from the beads by incubating at 50°C on a thermomixer with 250 μL 1X DNA IP buffer and 4 μL proteinase K (20 μg/μL) for 3 hours. DNA from IP samples were cleaned through PCI extraction and ethanol precipitation. DNA was PCR amplified with KAPA HiFi polymerase using 16 cycles of PCR. Each library was size-selected on a 1% agarose gel, its concentration determined using a Qubit (Thermo), and the integrity was confirmed by Topo-cloning a portion and sequencing ~10 fragments from each library. Libraries were sequenced on an Illumina HiSeq2000 using single-end sequencing at the UMass Medical School deep sequencing core facility. Resulting fastq sequences were processed and analyzed using the HOMER software.
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| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
hMeDIP-Seq
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| Data processing |
Single-end fastq reads were split by barcode adapter sequences, adapter sequences were removed, and reads were mapped to the mm9 genome using bowtie, allowing up to three mismatches. Aligned reads were processed in HOMER (Heinz et al., 2010). Genome browser tracks (bedGraph files) were generated from mapped reads using the “makeUCSCfile” command. Mapped reads were aligned over specific regions using the “annotatePeaks” command to make 20 bp bins over regions of interest and sum the reads within each window. Experiments were aligned over the following datasets: TSS reference sites (from HOMER software), intermediate CpG promoters (ICPs, defined in (Weber et al., 2007)), low methylated regions (LMRs, from (Stadler et al., 2011)), DNaseI hypersensitive sites (DHSs) from mouse ENCODE data (GSM1014154) with TSS locations removed, Mbd3 peaks (peaks called from both EGFP KD Mbd3 ChIP-seq libraries using HOMER) and Mbd2 peaks (peaks called from both EGFP KD Mbd2 ChIP-seq libraries using HOMER). After anchoring data over reference sites, aggregation plots were generated by averaging data obtained from biological replicates.
Genome_build: mm9
Supplementary_files_format_and_content: bedGraph
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| Submission date |
Mar 31, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Sarah J Hainer |
| E-mail(s) |
[email protected]
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| Organization name |
University of Pittsburgh
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| Department |
Department of Biological Sciecnes
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| Lab |
Hainer Lab
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| Street address |
4249 Fifth Ave
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15260 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE79770 |
DNA methylation is required for chromatin binding by Mbd2 and Mbd3 in ES cells (ChIP-Seq, meDIP-seq and hmDIP-seq) |
| GSE79771 |
DNA methylation is required for chromatin binding by Mbd2 and Mbd3 in ES cells |
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| Relations |
| BioSample |
SAMN04593870 |
| SRA |
SRX1671558 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2102275_ESC_Dnmt1KD_hmDIP_rep1.ucsc.bedGraph.gz |
138.9 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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