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| Status |
Public on Jun 18, 2019 |
| Title |
iPSCs_No13_methylseq |
| Sample type |
SRA |
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| Source name |
iPSCs
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| Organism |
Mus musculus |
| Characteristics |
tissue: iPSCs
passage: p4
strain: F1 (129X1/SvJ and MSM/Ms)
gender: Male
analysis method: Methylseq
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| Growth protocol |
MEFs were isolated from E14.5 embryos and were cultured in DMEM (Nacalai tesque) with 2 mM L-glutamine (Nacalai tesque), 100× nonessential amino acids (NEAA) (Nacalai tesque), 100 U/ml penicillin, 100 mg/ml streptomycin (P/S) (Nacalai tesque) and 10% Fetal Bovine Serum (FBS) (GIBCO). ESCs and iPSCs are maintained on feeders (mitomycin C (Kyowa Hakko Kirin Co., Ltd.)-treated MEFs) in knockout DMEM (GIBCO) with 2 mM L-glutamine, 100×NEAA, 100 U/ml penicillin, 100 g/ml streptomycin (P/S), 15% Fetal Bovine Serum (FBS), 0.11 mM β-mercaptoethanol (GIBCO) and 1000 U/ml human recombinant leukemia inhibitory factor (LIF) (Wako). EpiSCs were cultured in DMEM/F12+L-Glu (GIBCO), NEAA, P/S, 0.11 mM -mercaptoethanol, 20% KnockOut Serum Replacement (KSR) (GIBCO), which was supplemented with 10 ng/ml Human Recombinant Fibroblast Growth Factor (basic) (rh bFGF) (wako), and 10 M Y-27632 (wako). iEpiSCs were maintained in DMEM/F12+L-Glu supplemented with B27 (GIBCO) and N2 cell-supplements (GIBCO), NEAA, P/S, 0.11 mM β-mercaptoethanol, 15% FBS, which was supplemented with 10 ng/ml rh bFGF, 20 ng/ml Recombinant Activin A (R&D), 200 ng/ml anti-LIF antibody (Funakoshi).
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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) or NucleoSpin RNA plus (MACHEREY-NAGEL) 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). 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 |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Libraries were sequenced on the HiSeq2500 (2×100 bp paired-end reads, illumina) or Nextseq500 100 bp, single read, illumina)
Low quality bases and adapters in sequenced reads were trimmed with cutadapt-1.9.1
For 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.
Supplementary_files_format_and_content: ProcessedDataMatrixRPKMfin.STAR.txt:Tab-delimited text file includes RPKMs for each sample.
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| Submission date |
Feb 27, 2018 |
| Last update date |
Jun 18, 2019 |
| Contact name |
Yasuhiro Yamada |
| E-mail(s) |
[email protected]
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| Organization name |
University of Tokyo
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| Department |
Department of Molecular Pathology
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| Street address |
7-3-1 Hongo, Bunkyo-ku
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| City |
Tokyo |
| ZIP/Postal code |
113-0033 |
| Country |
Japan |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE111173 |
De novo DNA methylation at imprinted loci during reprogramming into naïve and primed pluripotency |
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| Relations |
| BioSample |
SAMN08616330 |
| SRA |
SRX3746050 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3024639_iPS_13.bedGraph.gz |
46.3 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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