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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 15, 2014 |
| Title |
Mus_Musculus - Cerebellum, H3K27ac ChIP |
| Sample type |
SRA |
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| Source name |
Cerebellum
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| Organism |
Homo sapiens |
| Characteristics |
chip antibody: H3K27ac (Abcam, ab4729)
tissue: Cerebellum
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Frozen tissues were dissected at -20ºC, thawed quickly and homogenized. Between 20mm3 and 0.5cm3 was used depending on tissue availability. Tissue was immediately chemically cross-linked by the addition of 2ml one-fifth volume of fresh 11% formaldehyde solution in medium and incubated for 10 minutes while rotating at room temperature. Cells were washed with PBS at 4ºC.Cells were lysed and sonicated. The IP was incubated overnight at 4°C with 50 ul of Dynal Protein G magnetic beads (Invitrogen lot# 90513840) that had been pre-incubated with 5 µg of the appropriate antibody for at least 3 hrs in PBS/0.5% BSA at 4°C. Beads were washed 4 times with RIPA buffer and 1 time with TE containing 50 mM NaCl. Bound complexes were eluted from the beads in elution buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS) and at the same time crosslinking was reversed by overnight incubation at 65°C. After removal of the beads immunoprecipitated DNA was diluted 1:1 with TE and then treated with RNAse A for 2 hours at 37°C, followed by proteinase K treatment for 2 hours at 50°C. DNA was purified using two consecutive phenol:chloroform extractions using MaXtract High Density gel tubes. The resulting DNA was used for analysis either on the solexa sequencer (HiSeq 2000 genome sequencer) or the SOLiD sequencer (5500xl /550xlW). The sample prep was preformed by using the sequencer corresponding ChIP-Seq sample prep kit according to the manufacturers protocol. HiSeq samples were prepped and run by the MIT BioMicro Center (http://openwetware.org/wiki/BioMicroCenter) using the HiSeq 2000 genome sequencer (10 ChIPs multiplexed per lane). Solexa samples were prepped and run in-house using the SOLiD 5500xl or SOLiD 5500xlW.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Human: Solexa sequencing: ChIP seq reads were mapped with bowtie 0.12.7 with a seed (-l) as long as the read, allowing 1 mismatch(-n) and only 1 alignment(-m) per seed. Best singleton alignment were given (- - best). SOLiD sequencing: ChIP seq reads were mapped with bowtie 0.12.7 with a seed (-l) of 36bp, allowing 3 mismatch(-n) and only 1 alignment(-m) per seed. Best singleton alignment were given (- - best).
Enriched Regions were determent by MACS2 (version2.0.8) with a threshold of 1e-5 (-p), a shiftsize of 200 (--nomodel,--shiftsize), and a large lambda of 100000 (--llocal).
Mice: ChIP seq reads were mapped with bowtie 0.12.7 with a seed (-l) as long as the read, allowing 1 mismatch(-n) and only 1 alignment(-m) per seed. Best singleton alignment were given (- - best).
Enriched Regions were determent by MACS2 (version2.0.8) with a threshold of 1e-5 (-p).
Rat: ChIP seq reads were mapped with bowtie 0.12.7 with a seed (-l) of 36bp, allowing 3 mismatch(-n) and only 1 alignment(-m) per seed. Best singleton alignment were given (- - best).
Enriched Regions were determent by MACS2 (version2.0.8) with a threshold of 1e-5 (-p), a shiftsize of 200 (--nomodel,--shiftsize), and a large lambda of 100000 (--llocal).
genome build: hg19, mm9, rn4
Supplementary_files_format_and_content: bedgraph: Treat pileup MACS2 bedGraph format.
Supplementary_files_format_and_content: bw: Treat pileup in bigWig format, read per million normalized.
Supplementary_files_format_and_content: peaks.txt: Peaks in MACS2 txt format.
Supplementary_files_format_and_content: peaks.bed: Peaks in MACS2 BED format.
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| Submission date |
Apr 09, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Menno P Creyghton |
| E-mail(s) |
[email protected]
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| Organization name |
Hubrecht Institute
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| Lab |
Creyghton
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| Street address |
Uppsalalaan 8
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| City |
Utrecht |
| ZIP/Postal code |
3584 CT |
| Country |
Netherlands |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE40465 |
Large-Scale Identification of Coregulated Enhancer Networks in the Adult Human Brain |
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| Relations |
| Reanalyzed by |
GSM1660062 |
| SRA |
SRX263385 |
| BioSample |
SAMN02356062 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1119262_Mouse_Cerebellum_H3K27ac_mm9.bw |
240.5 Mb |
(ftp)(http) |
BW |
| GSM1119262_Mouse_Cerebellum_H3K27ac_mm9_peaks.bed.gz |
610.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data not applicable for this record |
| Raw data are available in SRA |
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