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| Status |
Public on Jul 27, 2017 |
| Title |
t2i_XX_1_p15_WGBS |
| Sample type |
SRA |
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| Source name |
ES cells
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| Organism |
Mus musculus |
| Characteristics |
passage: p15
strain: F1 (129X1/SvJ and C57BL6)
cell type: ES cells
gender: female
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
PureLink Genomic DNA mini kit (invitrogen) for genomic DNA and RNAeasy plus Mini Kit (QIAGEN) for RNA.
For methyl-seq library preparation, 3μg of genomic DNA was sheared by covaris. Libraries were constructed using SureSelectXT Mouse Methyl-Seq Reagent Kit (Agilent Technologies). Bisulfite treatment was perfomed by EZ DNA methylation-GOLD kit (ZYMO RESEARCH). For whole genome bisulfite sequencing library, 500ng of DNA was sheared by covaris and ligated with methylated adapters supplied by TruSeqTM DNA, Sample Prep Kit-v2 (Illumina). Subsequently, DNA was bisulfite-treated using EZ DNA Methylation-Gold KitTM according to the supplier’s instruction. Final library amplification was performed using Pfu Turbo Cx (Agilent Technologies). The libraries were then sequenced on HiSeq 2500 (2 X 101 bp paired-end reads, Illumina). To generate RNA-seq libraries, Truseq Stranded mRNA LT sample prep kit (Illumina) was used according to the supplier’s instruction.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Libraries were sequenced on the HiSeq2500 (2 X 101 bp or 2×100 bp paired-end reads, illumina) or Nextseq500 (75 bp, single read, illumina)
Low quality bases and adapters in sequenced reads were trimmed with cutadapt-1.9.1
For normal WGBS analysis, the trimmed reads were mapped to the mouse genome (mm10) by Bismark software-v0.15.0 with bowtie2 (version 2.2.8) and default settings, and the mapped reads with high mapping quality (MAPQ>=20) were used for extraction of methylated cytosines by the Bismark methylation extractor with --ignore 10 --ignore_3prime 5 options (single-end reads) or with --ignore 10 --ignore_r2 10 --ignore_3prime 5 --ignore_3prime_r2 5 options (paired-end reads).
For allelic methylation analysis, the trimmed reads were mapped to both B6 mouse genome (mm10) and MSM/ms mouse genome independently by Bismark software-v0.15.0 with bowtie2 (version 2.2.8) and default settings, and the reads mapped to the same chromosome and positions of both B6 and MSM/ms genomes with high mapping quality (MAPQ>=20) were used for further analysis. MSM/ms mouse genome were reconstructed from mm10 using the SNPs (NIG Mouse Genome Database (MSMv4HQ, http://molossinus.lab.nig.ac.jp/msmdb/index.jsp)). Chromosomes Y and M were ommited from MSM/ms genomes because of lack of the SNPs information.
For caluculation of allelic methylation levels, the B6-derived and MSM/ms-derived sequenced reads were selected based on the MSM/ms SNPs data which were not in CpG sites and the patterns of bisulfite conversion. Methylated cytosines were extracted from the reads by the Bismark methylation extractor with --ignore 10 --ignore_r2 10 --ignore_3prime 5 --ignore_3prime_r2 5 options. For allelic methylation analysis with methyl-seq libraries, only the original bottom (OB) data were used for analysis of methylation status and methylation percentages were represented at the sites which have equal or more than 5 read depth.
For RNA-seq analysis, the sequenced reads were mapped to the mouse reference genome (mm10) using tophat-2.1.0 11 with the GENCODE version M9 annotation gtf file and the aligner Bowtie2-2.2.5 6 after trimming adaptor sequences and low-quality bases by cutadapt-1.9.1 12. For allelic expression analysis of RNA-seq, the trimmed reads were also mapped to MSM genomes described as above, and the reads mapped to the same chromosome and positions of both B6 and MSM/Ms genomes with high mapping quality (MAPQ>=20) were used for further analyses. The expression level of each gene was calculated as reads per kilobase per million mapped reads (RPKM) by cufflinks-2.2.1
Genome_build: mm10
Supplementary_files_format_and_content: Supplementary_files_format_and_content: bedGraph colums: <chromosome> <start position> <end position> <methylation percentage>, *The coordinates are zero-based, half-open.
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| Submission date |
Apr 10, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Yasuhiro Yamada |
| E-mail(s) |
yasu@ims.u-tokyo.ac.jp
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| Organization name |
University of Tokyo
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| Department |
Institute of Medical Science
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| Lab |
Division of Stem Cell Pathology
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| Street address |
4-6-1, Shirokanedai, Minato-ku
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| City |
Tokyo |
| ZIP/Postal code |
108-8639 |
| Country |
Japan |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE84164 |
Derivation of ground-state female ESCs maintaining gamete-derived DNA methylation |
| GSE84165 |
Derivation of ground-state female ESCs maintaining gamete-derived DNA methylation |
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| Relations |
| BioSample |
SAMN06704893 |
| SRA |
SRX2731761 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2572321_t2i_XX_1_p15.bedGraph.gz |
22.1 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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