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| Status |
Public on Nov 26, 2019 |
| Title |
MeCP2_WT_H3K36me3_ChIPseq_rep5 |
| Sample type |
SRA |
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| Source name |
Forebrain tissue
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: 8 weeks
gender: Male
genotype: WT
chip antibody: H3K36me3 (Abcam ab9050)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Forebrain (cortex and hippocampus) was dissected from 8-to-12-week-old male mice and flash frozen. Tissue was homogenized in 1% formaldehyde and cross-linked for 10 minutes at room temperature. Cross-linking was quenched with 125 mM glycine for 5 minutes at room temperature. Tissue was pelleted by spinning at 500 g for 5 minutes at 4°C, washed once with PBS, then spun again at 500 g for 5 minutes at 4°C. Cells were lysed by resuspending in L1 buffer (50 mM Hepes pH 7.5, 140mM NaCl, 1 mM EDTA pH 8, 1 mM EGTA pH 8, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, protease inhibitors) and rotating at 4°C for 10 minutes, then spun at 500 g for 5 minutes at 4°C. Nuclei were washed for 10 minutes at 4°C in L2 buffer (10 mM Tris pH 8, 200 mM NaCl, protease inhibitors), then spun at 500 g for 5 minutes at 4°C. Nuclei were resuspended in LB3 buffer (10 mM Tris pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-Lauroylsarcosine, protease inhibitors), and sonicated in a Diagenode Bioruptor (High Power, 60 cycles, 30 seconds on/45 seconds off). Insoluble material was removed by spinning at 16,000 g for 10 minutes at 4°C, and Triton X-100 was added to soluble chromatin at a final concentration of 1%. Chromatin was pre-cleared for two hours with Protein A (for rabbit antibodies) or Protein G (for mouse antibodies) Dynabeads, then incubated with Protein A or Protein G Dynabeads conjugated to antibodies overnight at 4°C. Antibodies used were: H3K27ac (Abcam ab4729), H3K36me3 (Abcam ab9050), H4K12ac (Millipore 07-595), H3K9ac (Millipore 06-942), H3K79me2 (Abcam ab3594), Pol II (Abcam ab817), Pol II Ser5p (Millipore 04-1572), and MeCP2 (Chen et al., 2003). Beads were washed twice with Low Salt Buffer (20 mM Tris pH 8, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), twice with High Salt Buffer (20 mM Tris pH 8, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), twice with LiCl Wash Buffer (10 mM Tris pH 8, 1 mM EDTA, 1% NP-40, 250 mM LiCl, 1% sodium deoxycholate) and once with TE Buffer (50 mM Tris pH 8, 10 mM EDTA) at 4°C. Chromatin was eluted off beads by incubating in TE Buffer with 1% SDS at 65°C for one hour, and crosslinks were reversed by incubating overnight at 65°C. Chromatin was treated with RNase A for 30 minutes at 37°C and Proteinase K for 2 hours at 55°C. DNA was phenol-chloroform extracted and purified with the Qiagen PCR purification kit.
Libraries were generated using the NuGEN Ovation Ultralow System V2 following manufacturer instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
10WT_10MeCP2_KO_H3K36me3_ChIPseq_input_gene_body_counts.txt
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| Data processing |
Sequencing reads were trimmed with Trimmomatic (v0.36) to remove adaptors and low quality sequence (settings: LEADING:5 TRAILING:5 SLIDINGWINDOW:4:20 MINLEN:50).
Trimmed reads were mapped to the mm10 genome with Bowtie2 (v2.2.9) with default parameters.
PCR duplicate reads were removed with Samtools (v0.1.19) rmdup.
Reads were extended to 250 bp (to approximate fragment length) with UCSC tools bedextendranges.
For each ChIP antibody, reads for each sample were randomly down-sampled to equal numbers using the GNU shuf utility.
ChIP-seq reads were quantified in the genomic regions specified in figure legends using Bedtools map (v2.26).
Genome_build: mm10
Supplementary_files_format_and_content: Tables of raw ChIP read counts in either full gene bodies (transcription start site (TSS) to transcription termination site (TTS) of longest isoform), gene bodies excluding the first 3kb (TSS +3kb to TTS), or TSS (TSS -1kb to +1kb). For YY1 ChIP, file contains position of YY1 motifs in YY1 peaks.
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| Submission date |
Mar 12, 2019 |
| Last update date |
Nov 26, 2019 |
| Contact name |
Lisa D. Boxer |
| E-mail(s) |
lisa_boxer@hms.harvard.edu
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| Organization name |
Harvard Medical School
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| Department |
Neurobiology
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| Lab |
Michael Greenberg
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| Street address |
220 Longwood Ave., Goldenson Bldg Rm 405
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE128182 |
MeCP2 represses the rate of transcriptional initiation of highly methylated long genes (ChIP-Seq I) |
| GSE128186 |
MeCP2 represses the rate of transcriptional initiation of highly methylated long genes |
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| Relations |
| BioSample |
SAMN11106875 |
| SRA |
SRX5508942 |
| 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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