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| Status |
Public on Oct 26, 2012 |
| Title |
MethylCap-seq_480N |
| Sample type |
SRA |
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| Source name |
normalcolontissue
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| Organism |
Homo sapiens |
| Characteristics |
tissue: normal colon tissue
technique: MethylCap-sequencing
starting molecule: genomic DNA
antibody/capture: Protein domain: His6–GST–MBD (Diagenode)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
MethylCap (Capturing of methylated DNA) was done using in automated procedure using a SX-8G/IP-StarTM robot (Diagenode). 2 µg of His6–GST–MBD (Diagenode) was incubated with 1 µg of fragmented DNA in binding buffer (BB, 20 mM Tris–HCl pH 8.5, 0.1% Triton X-100) with 200 mM NaCl in a final volume of 200 µl. This solution is mixed at 4 °C for 2 h. Magnetic GST-beads were prepared by washing 35 µl of a well-mixed beads suspension (Promega) with 200 µl of binding buffer + 200 mM NaCl at 4 °C. The suspension was mixed and a magnet was used to collect the beads. After removal of the supernatant the procedure is repeated once. After the second wash the supernatant is removed, but the beads are not allowed to get dry. The GST–MBD solution is added to the washed beads and this suspension is mixed for another hour at 4°C. After removal of the supernatant (the ‘flow-through’) the beads–GST–MBD–DNA complexes are washed/eluted. 200 µl of binding buffer with increasing concentrations of NaCl is added and the suspension is rotated for 10 min at 4 °C. Beads are captured on the magnet, and the supernatant is collected. Three different eluates were collected (LOW, MEDIUM, HIGH), corresponding 500 mM, 600 mM and 800 mM NaCl. For MethylCap-seq the MEDIUM and HIGH eluates were used separately.
1 to 30 ng of DNA was used for library preparation using the ChIP-seq DNA sample Prep Kit (Illumina, cat# IP-102–1001) using the recommended protocol. After ligation of adapters, a 300 bp band is excised using the E-Gel system (Invitrogen), which correspond to a 234 bp fragment of genomic DNA. PCR enrichment of adapter-ligated DNA was done using 14 to 16 cycles of PCR.
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| Library strategy |
MBD-Seq |
| Library source |
genomic |
| Library selection |
MBD2 protein methyl-CpG binding domain |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Cluster generation and sequencing-by-synthesis (36 bp) was performed using the Illumina Genome Analyzer IIx (GAIIx) platform according to standard protocols of the manufacturer (Illumina). The image files generated by the Genome Analyzer were processed to extract DNA sequence data. Sequence reads were aligned to the human (hg18) reference genome using the Illumina Analysis Pipeline allowing, one mismatch. Only the 36 bp sequence reads uniquely aligning to the genome were considered for further analysis. The output data were converted to Browser Extensible Data (BED) files for downstream analysis and Wiggle (WIG) files for viewing the data in the UCSC Genome Browser. All sequence analyses were conducted based on the Homo Sapiens hg18 genome assembly.
Genome_build: hg18
Supplementary_files_format_and_content: The file "MergedPeaks_normalized_tagdensity.gz" contains processed data for 24 patient sample pairs. It is a tab-delimited text file. MethylCap-seq was performed on 24 micro-dissected tumors and an equal number of matched microscopically normal tissue samples (48 DNA methylation profiles in total). Enriched regions (peaks) were called for each sample on the basis of a Poisson distribution of overlapping sequence reads within a dynamic window. A false discovery rate (FDR) was calculated relative to the total covered sequence, and peaks with an FDR of ≤10-6 were selected. All peaks from the 48 samples were merged. Per sample the number of tags overlapping each region of interest was calculated and normalized by dividing by the total number of aligned reads of the sample, and the length of the peak.
Supplementary_files_format_and_content: The file "MergedPeaks_normalized_tagdensity_add_5_samples.gz" contains processed data for 5 patient sample pairs. It is a tab-delimited text file. MethylCap-seq was performed on 5 tumors and an equal number of matched normal tissue samples. The peaks identified with the larger sample set was used, per sample the number of tags overlapping each region of interest was calculated and normalized by dividing by the total number of aligned reads of the sample, and the length of the peak.
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| Submission date |
Sep 14, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Arjen Brinkman |
| E-mail(s) |
arjen.brinkman@gmail.com
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| Organization name |
Radboud University, Nijmegen Center for Molecular Life Sciences
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| Department |
Molecular Biology
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| Lab |
Stunnenberg
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| Street address |
NCMLS #274, Geert Grooteplein Zuid 30
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| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
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| Platform ID |
GPL10999 |
| Series (1) |
| GSE39068 |
Comparative genome-wide DNA methylation analysis of colorectal tumor and matched normal tissues |
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| Relations |
| SRA |
SRX186954 |
| BioSample |
SAMN01174514 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1004073_480N.reads.bed.gz |
174.6 Mb |
(ftp)(http) |
BED |
| GSM1004073_480N.wig.gz |
49.8 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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