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| Status |
Public on Nov 26, 2019 |
| Title |
WTTG_Input_Tg1 |
| Sample type |
SRA |
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| Source name |
Cortex
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| Organism |
Mus musculus |
| Characteristics |
genotype: MeCP2 overexpression
Sex: Male
strain: C57BL/6J
age: 7-10 weeks
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| Growth protocol |
Brain tissue was taken from MeCP2 knockout, overexpression and wild-type mice maintained under standard housing conditions.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cerebral cortex was dissected on ice in phosphate buffered saline from 1) MeCP2 knockout and wild-type male litter mates at 7-8 weeks old and 2) MeCP2 overexpression and wild-type male litter mates at 7-10 weeks old. The tissue was flash-frozen in liquid nitrogen and stored at -80°C. ChIP experiments were performed on half a cortex as previously described (Cohen et al., 2011), using an alternative chromatin fragmentation method. Chromatin were fragmented with Covaris E220 sonicator (5% Duty Factory, 140 Peak Incidence Power, 200 cycles per burst, milliTUBE 1mL AFA Fiber). ChIP was performed with H3K27ac (Abcam ab4729), H3K4me3 (Abcam ab1012), H3K36me3 (Active Motif 61101), and MeCP2 (rabbit polyclonal (Chen et al., 2003)).
ChIP libraries for H3K27ac, H3K4me3, and H3K36me3 were generated using Ovation Ultralow Library System V2 (NuGEN) and libraries for MeCP2 with Accel-NGS 2S Plus DNA Library Kit (Swift Biosciences).
Libraries were pooled to a final concentration of 8-10nM and sequenced using Illumina HiSeq 3000 with GTAC, yielding 15-20 million single-end reads per sample.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Sequenced reads were mapped to the mm9 genome using bowtie2 alignment, and reads were deduplicated to consolidate PCR duplicate reads. Deduplicated reads were used to quantify read density normalized by the number of reads per sample and by read length in basepairs. Bedtools coverage –counts was used to quantify ChIP signal at the transcriptional start site (TSS), gene body (GB), and transcriptional end site (TES). For consistency with methylation analysis, the TSS was defined as a 1kb region surrounding the TSS (+/-500bp), the GB was defined as 3kb downstream of the TSS to the end of the transcript, and the TES was defined as 2kb upstream through 3kb downstream of the end of the transcript. edgeR was then used to determine differential ChIP-signal across genotypes.
Genome_build: mm9
Supplementary_files_format_and_content: Tab-separated table (with header) of H3K27ac peaks, identifiers, and various peak-associated ChIP signals, and methylation information
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| Submission date |
Dec 05, 2018 |
| Last update date |
Nov 26, 2019 |
| Contact name |
Harrison Wren Gabel |
| E-mail(s) |
gabelh@pcg.wustl.edu
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| Organization name |
Harvard Medical School
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| Department |
Neurbiology
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| Lab |
Michael Greenberg
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| Street address |
220 longwood avenue
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| City |
brookline |
| State/province |
Massachusetts |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL21493 |
| Series (2) |
| GSE123371 |
Chromosome topology shapes neuronal non-CG DNA methylation to influence MeCP2-mediated enhancer repression (ChIP-Seq) |
| GSE123373 |
Chromosome topology shapes neuronal non-CG DNA methylation to influence MeCP2-mediated enhancer repression |
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| Relations |
| BioSample |
SAMN10524622 |
| SRA |
SRX5098361 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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