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| Status |
Public on Jul 12, 2013 |
| Title |
DNA_Methylation_E14_adapted_2i |
| Sample type |
SRA |
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| Source name |
Embryonic Stem cells
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| Organism |
Mus musculus |
| Characteristics |
line: E14
strain: 129/Ola
cell type: ES-cells
measurement: DNA methylation
growth: adapted_2i
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| Treatment protocol |
The 2i adapted cells were released from serum and LIF and cultured on 2i and LIF for at least 8 passages
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| Growth protocol |
Embryonic stem cells were cultured without feeders in the presence of leukaemia inhibitory factor (LIF) either in medium containing 10% foetal calf serum or in serum-free medium containing the MEK inhibitor PD0325901 (1mM) and the Gsk3 inhibitor CH99021 (3mM), together known as 2i. Rex-GFP cells are heterozygous for knock-in of a destabalized form of GFP to the Rex gene.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Pelleted ES cells were resuspended in 5 ml digestion buffer (100mM NaCl, 10mM Tris-HCl pH8, 15mM EDTA pH8, 0.5% SDS, 0.1mg/ml Proteinase K) per collected confluent 10 cm plate, and incubated at 55°C for 12-18 hr. DNA was isolated by two times extraction with 5 ml phenol/chloroform/isoamyl alcohol, and the aqueous top layer was transferred to a new tube. To precipitate DNA, 1/2 volume 7.5M ammonium acetate and 2 volumes 100% ethanol were added. After spinning, the supernatant was discarded, the DNA pellet washed with 70% ethanol, dried, and resuspended in water.
Library construction was performed according to Kulis et al. (2012; Nature Genetics). In short: We spiked genomic DNA (1 or 2 μg) with unmethylated λ DNA (5 ng of λ DNA per μg of genomic DNA) (Promega). We sheared DNA by sonication to 50–500 bp with a Covaris E220 and selected 150- to 300-bp fragments using AMPure XP beads (Agencourt Bioscience Corp.). We constructed genomic DNA libraries using the TruSeq Sample Preparation kit (Illumina Inc.) following the Illumina standard protocol: the ends of the fragments were repaired, an adenine was added at the 3′ extremities of the fragments and Illumina TruSeq adapters were ligated at each extremity. After adaptor ligation, we treated DNA with sodium bisulfite using the EpiTect Bisulfite kit (Qiagen) following the manufacturer’s instructions for formalin-fixed and paraffin-embedded (FFPE) tissue samples. We performed two rounds of conversion to get >99% conversion. We enriched adaptor-ligated DNA through seven cycles of PCR using the PfuTurboCx Hotstart DNA polymerase (Stratagene). We monitored library quality using the Agilent 2100 BioAnalyzer (Agilent) and determined the concentration of viable sequencing fragments (molecules carrying adapters at both extremities) by quantitative PCR using the Library Quantification Kit from KAPA Biosystem. We performed paired-end DNA sequencing (two reads of 100 bp each) using the Illumina Hi-Seq 2000.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
FASTQ sequence files were aligned using RMAPBS (Smith et al., 2009; Smith et al., in prep) allowing a maximum of 10 mismatches for efficient mapping of reads containing bisulfite converted unmethylated cytosines. Cytosine methylation levels were determined using the standard new pipeline within RMAPBS (Methpipe; Andrew Smith, in prep).
In short, both mates of the paired-end sequencing were mapped separately by RMAPBS. If mated reads mapped within 300 bp, the reads were merged and included for analysis. Mated reads mapping with a distance of more than 300 bp were excluded from further analysis. Mated reads mapped within 300 bp, but mapping equally well on multiple positions on the genome, were discarded. If multiple mated reads mapped on exactly the same genomic coordinates, all but one (at random) were discarded. Symmetric cytosines (within a CpG context) on both forward and reverse strands were combined. Cytosine methylation level was called per individual C as: #C/(#C+#T).
Genome_build: mm9
Supplementary_files_format_and_content: Cytosine methylation levels within CpG context are present in the methcounts bedGraph files; Coverage over the cytosines within CpG context are present in the coverage bedGraph files. These files can be used as tables, but can also be directly uploaded in the UCSC genome browser.
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| Submission date |
Oct 30, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Hendrik Marks |
| E-mail(s) |
h.marks@ncmls.ru.nl
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| Organization name |
Radboud University Nijmegen, RIMLS
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| Department |
Molecular Biology
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| Street address |
Geert Grooteplein 26/28
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| City |
Nijmegen |
| ZIP/Postal code |
6525GA |
| Country |
Netherlands |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE41923 |
Whole-genome bisulfite sequencing of two distinct interconvertible DNA methylomes of mouse embryonic stem cells |
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| Relations |
| SRA |
SRX202088 |
| BioSample |
SAMN01797653 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1027572_DNA_CpG_coverage_E14_2i_adapted.bedGraph.gz |
137.0 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM1027572_DNA_CpG_methcounts_E14_2i_adapted.bedGraph.gz |
147.7 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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