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| Status |
Public on Dec 24, 2013 |
| Title |
ES_CTCF_Bis_rep2 |
| Sample type |
SRA |
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| Source name |
embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Embryonic Stem Cells (ES)
strain: Mixed (129-C57Bl/6)
genotype: wild type
chip antibody: anti-CTCF (SantaCruz #15914)
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| Growth protocol |
Wild type embryonic stem cells derived from mixed 129-C57Bl/6 background blastocysts, were cultivated on feeder cells or 0.2% gelatine coated dishes. ES cell growth medium consisted of DMEM (Invitrogen) supplemented with 15% foetal calf serum (Invitrogen), 1x non-essential amino acids (Invitrogen), 1mM L-glutamine, LIF and 0.001% beta-mercaptoethanol. Differentiation was performed as previously described (Bibel et al).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation (ChIP) assay for CTCF was performed according to the Upstate protocol using the antibody anti-CTCF (SantaCruz #15914) and starting from 70 μg of chromatin. Enrichments were checked by real time PCR using SYBR Green chemistry (ABI) and 1/80 of ChIP or 20ng of input chromatin per PCR reaction. DNA generated from 5 different ChIP experiments (≈100 ng) was pooled and used for subsequent library preparation. DNA fragments were end repaired by incubation at 20°C for 30 minutes with 400µM dNTP, 3 units of T4 DNA polymerase (NEB #M0203S), 5 units of DNA Polymerase I Lg. Frag. (Klenow) (NEB #M0210S), 10 units of T4 PNK (NEB #M0201S), 1x T4 DNA ligase buffer containing 10mM ATP (NEB), followed by column purification using QIAquick PCR Purification Kit (QIAGEN #28106). 3’ ends of DNA fragments were adenylated by incubation at 37°C for 30 minutes with 200µM dATP, 1xNEB Buffer 2, 5 units Klenow Fragment (3´->5´ exo–) (NEB # M0212L), followed by column purification using MinElute PCR Purification Kit (QIAGEN # 28006). Adapter for single end sequencing were reproduced based on Illumina adapter sequences (Oligonucleotide sequences © 2006-2008 Illumina, Inc. All rights reserved): 5’ P- GATXGGAAGAGXTXGTATGXXGTXTTXTGXTTG and 5’ AXAXTXTTTXXXTAXAXGAXGXTXTTXXGATXT, where X is a methylated cytosine. Adapters were ordered as single stranded oligos (Microsynth AG), resuspended in annealing buffer (10mM Tris pH7.5, 50mM NaCl, 1mM EDTA), annealed by heating at 95°C for 10 minutes and cooling down slowly. Annealed adapters were ligated to the DNA fragments by incubation at room temperature for 15 minutes in the following mix: 400 nM of annealed adapters, 1x NEB Quick ligase buffer, 2.000 units of T4 Quick ligase (NEB #M2200S), followed by column purification using MinElute PCR Purification Kit (QIAGEN #28006). 200 ng of Drosophila DNA (Kc cells) were then added as a carrier. Adapter-ligated DNA of 150-400 bp was selected from 2% agarose gel electrophoresis and purified using MinElute Gel Extraction Kit (QIAGEN #28606). BSA (final concentration 0.5μg/μl) was added to gel-purified DNA and the mix was then treated with sodium bisulfite using the Imprint® DNA Modification Kit (Sigma-Aldrich) as per manufacturer’s instructions. Bisulfite-converted, adapter-ligated DNA molecules were enriched using 18 cycles of PCR with the following reaction composition: 2.5 U of uracil-insensitive PfuTurboCx Hotstart DNA polymerase (Stratagene), 5 μl 10X PfuTurbo reaction buffer, 25 μM dNTPs, 0.5µM of Single End Illumina PCR primers (1.1 and 2.1). The thermocycling parameters were: 95°C 2 min, 98°C 30 sec, then 18 cycles of 98°C 15 sec, 65°C 30 sec and 72°C 3 min, ending with one 72°C 5 min step, and 4°C indefinitely, followed by column purification using the MinElute PCR Purification Kit (QIAGEN #28006). DNA was then run on 2% agarose gel electrophoresis to separate the library from adapter-adapter ligation products, and purified from the gel using the MinElute Gel Purification Kit (QIAGEN #28606). Quality of the libraries and template size distribution were assessed by running an aliquot of the library on an Agilent 2100 Bioanalyzer (Agilent Technologies).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
bisulfite converted
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| Data processing |
All C nucleotides in sequence reads from bisulfite converted samples were converted in silico to T nucleotides, and the converted reads were aligned to a similarly converted genome separately to each strand using the software bowtie (version 0.10.0.1)(Langmead et al., Genome Biol. 2009;10(3):212) with parameters --best --strata -v 3 --norc -a.
Only reads with a unique alignment in this reduced alphabet base-space were retained, and C nucleotides from the original reads and genome were reintroduced.
To eliminate effects caused by polymorphisms in our experimental system, C nucleotides that overlapped known SNPs between the reference C57BL/6J and the 129S5 strains were removed from further analysis based on the SNPs identified by the Mouse Genomes Project at Sanger Institute (downloaded from ftp://ftp-mouse.sanger.ac.uk/REL-1003/SNPs/20100301-all-snps.tab.gz).
Total and methylated counts for all covered CpGs in the genome were calculated as the number of alignments with either C (methylated) or T (unmethylated) and the number of alignments with C (methylated), combining the counts from the two Cs in a CpG and its reverse complement (position i on plus strand and position i+1 on minus strand).
Genome_build: mm9
Supplementary_files_format_and_content: Chromosome, Chromosomal position, Total number of reads, Number of methylated reads.
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| Submission date |
Sep 12, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Dirk Schuebeler |
| Organization name |
Friedrich Miescher Institute for Biomedical Research
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| Street address |
Maulbeerstrasse 66
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| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE39736 |
Transcription factor occupancy is linked to DNA methylation turnover at active regulatory regions [Bisulfite-Seq] |
| GSE39739 |
Transcription factor occupancy is linked to DNA methylation turnover at active regulatory regions |
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| Relations |
| BioSample |
SAMN02355978 |
| SRA |
SRX349421 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1230236_ES_CTCF_Bis_rep2.tsv.gz |
27.0 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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