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| Status |
Public on Oct 08, 2010 |
| Title |
SssI MBDCap-SF (lane 1) |
| Sample type |
SRA |
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| Source name |
SssI MBDCap-SF
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| Organism |
Homo sapiens |
| Characteristics |
cell type: M.Sssi-treated
mbdcap: SF
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| Growth protocol |
37 deg C with 5% CO2
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Sequencing libraries were prepared using the Chip-Seq DNA Sample Prep Kit (Illumina) according to the manufacturer's protocol
MBDCap: The MethylMiner™ Methylated DNA Enrichment Kit (Invitrogen) was used to isolate methylated DNA. 1 µg of genomic DNA was sonicated to 100-500 bps. 3.5 µg (7 µl) of MBD-Biotin Protein were coupled to 10 µl of Dynabeads M-280 Streptavidin according to the manufacturer’s instructions. The MBD-magnetic beads conjugates were washed 3 times and resuspended in 1 volume of 1 x Bind/Wash buffer. The capture reaction was performed by adding 1 µg sonicated DNA to the MBD-magnetic beads on a rotating mixer for 1 hour at room temperature. All capture reactions were done in duplicate. The beads were washed three times with 1 x Bind/Wash buffer. The methylated DNA was eluted in one of two ways: (1) as a single fraction with a High Salt Elution Buffer (2000mM NaCl), denoted MBD-SF, or (2) as distinct subpopulations based on the degree of methylation using an increasing NaCl concentration of the elution buffer, from 200mM to 2000mM in a stepwise gradient (Elution 1- 200mM, Elution 2 – 350mM, Elution 3 – 450mM, Elution 4 -600mM, Elution 5 -1000mM and Elution 6 – 2000mM). Each fraction was concentrated by ethanol precipitation using 1 µl glycogen (20 µg/µl), 1/10th volume of 3M sodium acetate, pH 5.2 and two sample volumes of 100% ethanol and resuspended in 60 µl H20.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
library strategy: MBDCap-Seq
library selection: MBDCap
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| Data processing |
Bowtie was used mapping, allowing 3 mismatches. Only reads mapping to unique genomic locations were kept. For the comparison to array data, read counts were generated using annotationCounts() in the Repitools package (Statham et al. 2010 Bioinformatics) and statistics for differential methylation were calculated using the edgeR package (Robinson et al. 2010 Bioinformatics).
The count tables from the sequencing data that were used to generate the comparative results to the array outputs are available on the Series record (TSS_counts.txt).
For comparison of array and sequencing data, the outputs of blocksStats() -- a procedure used for targeted analysis of differential methylation: blocksStats_diffMeth.txt supplementary file is provided on the Series record.
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| Submission date |
Oct 06, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Mark Robinson |
| E-mail(s) |
[email protected]
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| Organization name |
University of Zuerich
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| Department |
Institute of Molecular Life Sciences
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| Street address |
Winterthurerstrasse 190
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| City |
Zuerich |
| ZIP/Postal code |
8057 |
| Country |
Switzerland |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE24546 |
Evaluation of affinity-based genome-wide DNA methylation data: effects of CpG density, amplification bias and copy number variation |
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| Relations |
| SRA |
SRX028542 |
| BioSample |
SAMN00115736 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM605085.map.gz |
378.8 Mb |
(ftp)(http) |
MAP |
SRA Run Selector |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
| Raw data are available in SRA |
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