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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 01, 2017 |
| Title |
ESC.r1 (BS-Seq) |
| Sample type |
SRA |
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| Source name |
embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
strain: 129S2/SvPasCrl
cell type: embryonic stem cells
genotype/variation: wild type
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| Growth protocol |
Embryonic stem cells were cultured in different culture medium in vitro.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA (two biological replicates per condition) was extracted by QIAamp DNA Micro Kit (Qiagen).
100 ng of genomic DNA was subjected to 3.5 hours bisulfite treatment using the Methylcode Bisulfite Conversion Kit (Invitrogen). To monitor the bisulfite conversion efficiency, unmethylated Lambda DNA (0.5ng, Promega) was spiked-in before conversion. Single-stranded BS-converted DNA was subjected to the post-bisulfite adaptor tagging (PBAT) protocol as described previously. PBAT libraries were subjected to single-end 150 bp sequencing on HiSeq 4000 sequencing system (Illumina).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
PBAT-reads were quality-trimmed with TrimGalore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore), and 4 nt random primer sequences at the 5’ end and 1 nt at the 3’ end of all reads were removed. PBAT-reads were then mapped to the computationally bisulfite-converted mouse reference genome (GRCm38/mm10) by using Bismark (version: 0.16.3; parameter settings: ‘bismark --pbat -N 1 -L 40’). Potential PCR duplicates were removed using the deduplicate bismark command.
Post-alignment analysis was performed by using the MethPipe package. First, aligned sequences were converted to MethPipe format using the to-mr command. Methylation levels and coverage for each symmetric CpG site were calculated by the methcounts and symmetric-cpgs commands. Average CpG methylation levels of annotated genomic regions, i.e. promoters, exons, repeats, imprint control regions, CGIs, enhancers and 2 kb genomic tiles, were calculated with the MethPipe roimethstat program. Only high-confidence genomic regions with at least 40 CpG observations from reads in the region were used in further analyses.
2 kb tiles were calculated for all chromosomes with a 1 kb offset. Coordinates of promoters (-1000 bp and +100 bp from the transcription start site (TSS)), exons, introns and intergenic regions were retrieved from Ensembl (Mouse Release 86). CGI annotations were obtained from the UCSC Table Browser. Imprint control regions annotations were obtained from Tomizawa et al (2011). Tissue-specific enhancer coordinates (+/- 1000 bp from peak; mm9 coordinates were converted to mm10 by the UCSC liftOver tool) were retrieved from the Mouse Encode Project46. Hypermethylated regions and Hypomethylated regions were calculated by the hmr command from the MethPipe package.
Unsupervised hierarchical clustering of 2 kb tile methylation was performed using the R hclust command with the “ward” method. Correlation matrix was calculated by the R corrplot package. Profile plot was created by the R seqplots package. Gene ontology analysis was performed by GREAT and DAVID Bioinformatics Resource 6.8.
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: The methpipe CpG.meth files are tab delimited and contain methylation level at each CpG site in the following format: <chromosome> <C lcoation> <strand> <sequence context> <methylation level> <coverage>
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| Submission date |
May 31, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Sabine Dietmann |
| Organization name |
Washington University School of Medicine
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| Street address |
4565 McKinley Avenue
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| City |
St Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE99488 |
Enhancing the Potency of Mouse Embryonic Stem Cells [BS-Seq] |
| GSE99494 |
Enhancing the Potency of Mouse Embryonic Stem Cells |
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| Relations |
| BioSample |
SAMN07180087 |
| SRA |
SRX2871967 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2644747_PBAT.ESC.2iL.r1.meth.txt.gz |
93.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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