 |
 |
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Apr 29, 2015 |
Title |
S6KO Input |
Sample type |
SRA |
|
|
Source name |
mouse embryonic stem cells
|
Organism |
Mus musculus |
Characteristics |
strain: 129 genotype/variation: Sirt6 knockout sample type: input
|
Growth protocol |
WT and S6KO ES cells were grown in ES media conditions
|
Extracted molecule |
genomic DNA |
Extraction protocol |
WT and S6KO ES cells were harvested at approximately 80% - 90% confluence, washed with PBS and kept at -20°C Genomic DNA was sheared using Covaris Adaptive Focused Acoustic to the center at 200 bp. DNA fragments were size selected and ligated with methylated adaptors and treated with sodium bisulfite (invitrogen). The extra adapters were removed by AmpurXP (Beckman). The DNA was then denatured for 10 minutes at 95 °C (0.4 M NaOH, 10 mM EDTA), neutralized by the addition of cold 2M ammonium acetate (pH 7.0) for 10 min on ice. Then denatured DNA fragments were incubated with 1 μl anti-CMS antiserum in 1x IP buffer (10 mM sodium phosphate pH 7.0, 140 mM NaCl, 0.05% Triton X-100) for 2 h at 4 °C, and then precipitated with protein G beads. Precipitated DNA was eluted with proteinase K, purified with phenol chloroform, amplified and enriched by 8 cycles PCR using Pfu TurboCx hotstart DNA polymerase (Stratagene). DNA sequencing was carried out using Illumina HiSeq sequencing systems.
|
|
|
Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2000 |
|
|
Description |
Other: CMS-IP
|
Data processing |
Sequencing reads derived from S6KO and WT ESCs CMS-IP and Input assays were mapped against mm9 as previously described for bisulfite treated samples (Pastor et al., 2011, PMID: 21552279). After discarding all reads that map to multiple positions in the genome, there are 11,679,660 reads for S6KO CMS-IP, 13,916,523 reads for WT CMS-IP, 24,157,471 reads for S6KO input, and 16,743,260 reads for WT input. To identify genome wide differentially hydroxymethylated regions (DHMRs) in S6KO compared to WT, we calculated differential coverage at genome wide 500 bp windows by employing the Bioconductor package MEDIPS (Lienhard et al., 2014, PMID: 24227674) v1.12.0 (extend = 200, uniq = T, window_size = 500, BSgenome = BSgenome.Mmusculus.UCSC.mm9, adj = F, diff.method = edgeR, p.value = 1e-3), resulting in 1,562 genomic windows with significant loss of 5hmC in S6KO, and 2,218 genomic windows with significant gain of 5hmC. Genome_build: mm9 Supplementary_files_format_and_content: Result table contains counts, rpkm, log2FC, and p-values at genome wide 500 bp windows as returned by the MEDIPS software for significant DHMRs (p.value < 1e-3).
|
|
|
Submission date |
May 30, 2014 |
Last update date |
May 15, 2019 |
Contact name |
Kenneth N Ross |
E-mail(s) |
Kenneth.Ross@dfci.harvard.edu
|
Organization name |
Dana-Farber Cancer Institute
|
Department |
Pediatric Oncology
|
Street address |
450 Brookline Ave., Rm M640
|
City |
Boston |
State/province |
MA |
ZIP/Postal code |
02115 |
Country |
USA |
|
|
Platform ID |
GPL13112 |
Series (1) |
GSE65836 |
The Histone Deacetylase Sirt6 Controls Embryonic Stem Cell Fate Via Tet-Mediated Production of 5-Hydroxymethylcytosine |
|
Relations |
BioSample |
SAMN02808777 |
SRA |
SRX1101133 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
|
|
|
|
 |