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| Status |
Public on Dec 24, 2013 |
| Title |
RESTko_ES_Bis |
| Sample type |
SRA |
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| Source name |
REST knockout ES cells
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| Organism |
Mus musculus |
| Characteristics |
strain: Black Agouti 129
cell type: Embryonic Stem Cells (ES)
genotype/variation: REST knockout
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| Growth protocol |
REST KO embryonic stem cells derived from Black Agouti 129 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 |
The REST KO methylome library was prepared following Illumina Paired-End Sample Preparation Guide, with the following modifications: Upon adapter ligation and gel selection, purified DNA was converted with sodium bisulfite using the Imprint DNA Modification Kit (Sigma-Aldrich) as per manufacturer's instructions. One third of the bisulfite-converted, adapter-ligated DNA molecules were enriched by PCR with the following reaction composition: 2.5 U of uracil-insensitive PfuTurboCx Hotstart DNA polymerase (Stratagene), 5ul 10X PfuTurbo reaction buffer, 25uM dNTPs, 0.5uM of Illumina PE-PCR primers. The thermocycling parameters were: 95C 2 min, 98C 30 sec, then 10 cycles of 98C 15 sec, 65C 30 sec and 72C 3 min, ending with one 72C 5 min step. Amplified DNA was then purified using Agencourt AMPure XP beads (Beckman-Coulter). Quality of the libraries and template size distribution were assessed by running an aliquot of the library on an Agilent 2100 Bioanalyzer (Agilen 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 |
Apr 26, 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 |
SAMN02058388 |
| SRA |
SRX272068 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1129833_ES_RestKO_Bis.tsv.gz |
73.4 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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