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| Status |
Public on Jun 17, 2015 |
| Title |
ChIP-seq_input_excitatory_neurons_rep2 |
| Sample type |
SRA |
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| Source name |
Excitatory pyramidal neurons from mouse neocortex
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6J/129
genotype: Camk2a-cre; R26-LSL-CAG-Sun1-GFP-myc
tissue: brain (neocortex)
cell type: excitatory pyramidal neurons
age: 8 to 11 weeks
Sex: male
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| Growth protocol |
Adult mice were housed in our animal facility with 12 h light/dark cycles and food ab libitum. Animals were used for analysis in accordance with protocols approved by the Institutional Animal Care and Use Committee
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The neocortex was rapidly dissected from 1-2 adult mice on ice, and the INTACT method was used to affinity purify GFP+ nuclei. Nucleosomes for native ChIP-seq were prepared as previously described [Henry G.L. et al. Cell type-specific genomics of Drosophila neurons. Nucleic Acids Res. 40, 9691-9704 (2012)]. Briefly, 1-2 million bead-bound nuclei were digested with 0.025 units/uL micrococcal nuclease (Worthington LS004798) in 500uL of 15mM HEPES pH 7, 1mM KCl, 2mM MgCl2, 2mM CaCl2, 340mM sucrose, 0.15mM spermine, 0.5mM spermidine, and 5mM sodium butyrate at 37°C for 15 minutes. The reaction was terminated by the addition of EGTA to 2mM final concentration. Nucleosomes were extracted for 30 minutes on ice with 200uL 15mM HEPES pH7, 200mM NaCl, 25mM KCl, 2mM MgCl2, 1mM EGTA, 340mM sucrose, 0.15mM spermidine, 0.15mM spermine, and 5mM sodium butyrate. A second 30 minute extraction was performed with the same buffer except the salt concentration was raised to 400mM NaCl. The extracts were combined and dialyzed overnight against 15mM HEPES pH7, 25mM KCl, 1mM β-mercaptoethanol, 1mM PMSF, and 5mM sodium butyrate using a 10K cut-off Slide-a-Lyzer dialysis device (Thermo Scientific 88401).
Native ChIP and library construction were combined [Garber M. et al. A high-throughput chromatin immunoprecipitation approach reveals principles of dynamic gene regulation in mammals. Mol. Cell 47, 810-822 (2012); Henry G.L. et al. Cell type-specific genomics of Drosophila neurons. Nucleic Acids Res. 40, 9691-9704 (2012)]. Nucleosomes prepared from 0.5-1 million nuclei were incubated with 1ug antibody and 25uL Protein G Dynabeads. The following antibodies were used: rabbit anti-H3K27me3 (Millipore 07-449), rabbit anti-H3K27ac (Abcam ab4729), rabbit anti-H3K4me3 (Abcam ab8580), and rabbit anti-H3K4me1 (Abcam ab8895). Amplified nucleosomal cDNA was fragmented, end-repaired, linker adapted, and sequenced on an Illumina HiSeq 2500 for 50 cycles.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Data was mapped to the Mus musculus reference genome (mm10).
Analysis for RNA-seq, MethylC-seq, ATAC-seq, and ChIP-seq datasets were performed as described in Mo et al., 2014 (in submission)
ChIP-seq: Reads were aligned to the mm10 genome, keeping only uniquely aligning reads (BOWTIE v0.12.7 -m 1)
ChIP-seq: Visualization tracks were generated by counting genome-wide coverage of reads (BEDTOOLS v2.15.0 genomeCoverageBed -bg), scaling to 10M total alignments (custom perl script), and converting to bigWig format (UCSC wigToBigWig).
ChIP-seq: Peaks were called with SICER_V1.1 (redundancy threshold = 1; fragment size=150; W=200, G=200 for H3K27ac, H3K4me1, and H3K4me3; W=200, G=1000 for H3K27me3). Overlapping peaks from the 2 replicates were merged (BEDTOOLS merge) to generate 1 set of peaks for each histone modification.
sample_supplementary_files_format_and_content: bigWig tracks for visualization of RNA-seq, ATAC-seq, and ChIP-seq datasets for each histone modification.
sample_supplementary_files_format_and_content: tab delimited text files of ChIP-seq peaks; columns in peak files are: column 1 - chromosome; column 2 - start; column 3 - end
Genome_build: mm10
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| Submission date |
Nov 10, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Alisa Mo |
| E-mail(s) |
amo4@jhmi.edu
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| Phone |
410 955 4679
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| Organization name |
Johns Hopkins University School of Medicine
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| Department |
Molecular Biology and Genetics
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| Lab |
Jeremy Nathans
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| Street address |
725 N. Wolfe St. PCTB805
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE63137 |
Epigenomic Signatures of Neuronal Diversity in the Mammalian Brain |
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| Relations |
| BioSample |
SAMN03174739 |
| SRA |
SRX757087 |
| Named Annotation |
GSM1541979_ChIP-seq_input_excitatory_neurons_rep2_scaled10M.bw |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1541979_ChIP-seq_input_excitatory_neurons_rep2_scaled10M.bw |
192.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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