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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 09, 2014 |
| Title |
Ch12 MeDIP-seq |
| Sample type |
SRA |
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| Source name |
Ch12, mouse lymphoblast cell line
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| Organism |
Mus musculus |
| Characteristics |
cell line: Ch12
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For MeDIP, 5–15 μg DNA isolated using the DNeasy Tissue kit (Qiagen, Valencia, CA), than DNA was sonicated to ~100–500 bp with a Bioruptor sonicator (Diagenode). Sonicated DNA was end-repaired, A-tailed, and ligated to single-end adapters following the standard Illumina protocol. After agarose size-selection to remove unligated adapters, 2–5 μg of adaptor-ligated DNA was used for each immunoprecipitation using a mouse monoclonal anti-methylcytidine antibody (1 mg ml−1, Eurogentec. Catalog No. BI-MECY-0100). For this, DNA was heat-denatured at 95 °C for 10 min, rapidly cooled on ice, and immunoprecipitated with 1 μl primary antibody per microgram of DNA overnight at 4 °C with rocking agitation in 500 μl immunoprecipitation buffer (10 mM sodium phosphate buffer, pH 7.0, 140 mM NaCl, 0.05% Triton X-100). To recover the immunoabsorbed DNA fragments, 4 μl of rabbit anti-mouse IgG secondary antibody (2.5 mg ml−1, Jackson Immunoresearch. Catalog No. 315-005-008) and 100 μl Protein A/G beads (Pierce Biotechnology. Catalog No. PI20421) were added and incubated for an additional 2 h at 4 °C with agitation. After immunoprecipitation a total of six immunoprecipitation washes were performed with ice-cold immunoprecipitation buffer. Washed beads were resuspended in TE buffer with 0.25% SDS and 0.25 mg ml−1proteinase K for 2 h at 55 °C and then allowed to cool to room temperature. MeDIP DNA were purified using Qiagen MinElute columns and eluted in 16 μl EB (Qiagen).
Fifteen cycles of PCR were performed on 5 μl of the immunoprecipitated DNA using the single-end Illumina PCR primers. The resulting reactions were purified with Qiagen MinElute columns, after which a final size selection (220-520 bp) was performed by electrophoresis in DNA was eluted in 36 μl EB buffer using Qiagen QIAquick columns. After cleanup with Qiagen MinElute columns, each library was examined by spectrophotometry (Nanodrop, Thermo Scientific) and Agilent DNA Bioanalyzer (Agilent).2% agarose. Libraries were quality controlled by spectrophotometry and Agilent DNA Bioanalyzer analysis.
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| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina HiSeq 1000 |
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| Data processing |
Sequencing reads were aligned to human hg19 or mouse mm9 with BWA
For MeDIP-seq, redandunt reads were discard
For MRE-seq, reads were filtered by MRE recognization site in genome.
Aligned results were further processed by methylQA (http://methylqa.sourceforge.net/) to get bigWig files
Genome_build: hg19, mm9
Supplementary_files_format_and_content: bigWig
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| Submission date |
Apr 14, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Daofeng Li |
| E-mail(s) |
lidaof@gmail.com
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| Phone |
(314) 286-0866
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| Organization name |
Washington University in St. Louis
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| Department |
Genetics
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| Lab |
Ting Wang Lab
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| Street address |
4515 McKinley Avenue
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| City |
SAINT LOUIS |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platform ID |
GPL15103 |
| Series (1) |
| GSE57230 |
Widespread contribution of transposable elements to the innovation of gene regulatory networks [mouse ENCODE] |
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| Relations |
| BioSample |
SAMN02727256 |
| SRA |
SRX534888 |
| Named Annotation |
GSM1368909_TW248_Ch12_MeDIP.bigWig |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1368909_TW248_Ch12_MeDIP.bigWig |
358.8 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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