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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 10, 2017 |
| Title |
5mc_WT MeDIP-seq |
| Sample type |
SRA |
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| Source name |
mouse ES cells
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| Organism |
Mus musculus |
| Characteristics |
strain: 129SVxC57/BL6
cell type: cultured embryonic stem cells (ES cells)
genotype/variation: wild type
passages: passages 3-5
antibody: 5-Methylcytosine (5-mC) antibody (pAb); Active Motif Cat. #61255
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| Growth protocol |
Co-derived wild-type and Srap1 KO mouse ES cells were cultured on gelatin-coated plates in high-glucose DMEM containing 15% FBS, 1x non-essential amino acids, 2 mM L-glutamine, 100 mM beta-mercaptoethanol, 1x nucleosides (Millipore), 1000U/ml leukemia inhibitory factor (StemRD) and 2i (selective GSK3β & Mek 1/2 inhibitors; Millipore), 25 mg/ml L-ascorbic acid.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP, mouse ES cells (C57BL/6N x 129/Sv background) stably expressing FHA-Srap1 were fixed in 1% paraformaldehyde for 10 min at 37 °C, followed by quenching with 125 mM glycine for 5 min. Cells were lysed and processed using the MAGnify ChIP system (Life Sciences). Lysates were sonicated using an ultrasonic processor (Cole Parmer) at power setting 3 in 30 s pulses followed by 1 min on ice, for a total of 6 min sonication.
Nucleoprotein complexes were immunoprecipitated using a mixture of M2 Flag and HA antisera (2.5 mg each) or equivalent amounts of isotype-matched mouse IgG per sample.
For DIP experiments, genomic DNA was isolated from Srap1 KO and matched wild-type ES cells using the DNeasy Blood & Tissue DNA kit and sonicated for a total of 6 min.
Samples were processed using the MeDIP or hMeDIP kits (Active Motif) according to the manufacturer’s protocol. For 5fC/5caC DIP, the hMeDIP protocol was followed using polyclonal antibodies to 5fC or 5caC.
Library preparation was performed using the Kapa HyperPrep kit, followed by 12 cycles of PCR amplification with Kapa HiFi HotStart polymerase. Amplified libraries were pooled, purified twice with 0.9X Ampure XP beads and analyzed on the Illumina HiSeq2000 or NextSeq500 for single-end reads.
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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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| Data processing |
For ChIP- and DIP-seq, raw reads were trimmed to remove adapters using Trim Galore (v0.3.5) and Bowtie (v0.12.8) was used to obtain reads mapped uniquely to mouse genome (mm9).
The parameters used in Bowtie were ‘‘-v 3 -m 1 -y –best –strata’’. To accurately identify regions enriched for each cytosine modification, we first identified peak candidates with MACS (v2.1.1) (Ref. 31) using input as the control data set and using the following parameters: [–bw 300 -m 5,30–shiftsize 100 -p 1e-3–slocal = 1000 -B–keep-dup = 1].
To remove nonspecific signals, IgG samples were processed similarly and their normalized read density (reads per kilobase per million) values were subtracted from modification-specific peaks. The empirical FDR was estimated by exchanging the DIP and IgG control samples and identifying peaks in the control sample using the same set of parameters used for the DIP sample.
Called peaks were annotated with Gencode M1 and Cistrome CEAS32. Normalized read density plots centered on annotated elements downloaded from ENCODE were generated using NGSPLOT33. Peak overlap statistics were calculated using Fisherbed. All GO term analysis used DAVID (http://david.abcc.ncifcrf.gov).
For RRBS, adapter trimming for raw sequencing reads was performed using Trim Galore and reads with a Phred score of less than 20 were excluded. Trimmed reads were mapped against the reference mouse genome (mm10) with Bismark (v0.14.5)
CpGs with a minimal coverage of 8 were included in further analysis steps. A Chi-square test was used to identify significant differentially methylated regions in KO vs wild-type samples, and an FDR-adjusted P <0.01 was used to determine DMRs.
Genome_build: mm9
Supplementary_files_format_and_content: bed files were generated using
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| Submission date |
May 25, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Douglas Edmund Feldman |
| E-mail(s) |
defeldma@usc.edu
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| Organization name |
USC Keck School of Medicine
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| Department |
Pathology
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| Lab |
HMR 212
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| Street address |
2011 Zonal Avenue
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90033 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE81222 |
Erasure of Tet-oxidized 5-methylcytosine by a SRAP nuclease |
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| Relations |
| BioSample |
SAMN05176269 |
| SRA |
SRX1799798 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2176998_5mc_WT.bedGraph.gz |
10.3 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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