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| Status |
Public on Sep 22, 2017 |
| Title |
mOSN_H3K79me3_Mnase-ChIP |
| Sample type |
SRA |
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| Source name |
mature olfactory sensory neurons
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| Organism |
Mus musculus |
| Characteristics |
cell type: mature olfactory sensory neurons
genotype: OMP-IRES-GFP
chip antibody: anti-H3K79me3 (abcam, ab2621)
extract_protocol: Native-ChIP
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| Treatment protocol |
Mice were sacrificed using CO2 followed by cervical dislocation. The main olfactory epithelium (MOE) was dissected and transferred to ice-cold PBS. The MOE was cut in to small pieces with a razor blade, and then dissociated with a papain dissociation system (Worthington Biochemical). For horizontal basal cells, dissociated cells were resuspended in 800uL PBS with 1:80 CD54-PE antibody (BioLegend, Cat# 116107), incubated for 30 minutes at 4oC with rotation, and then washed twice with PBS. Labeled cells were then resuspended in sort media (PBS with 2% Fetal Bovine Serum) supplemented with 100 U/mL DNase I (Worthington Biochemical), 4mM MgCl2, and 500ng/mL DAPI (Invitrogen). For all other ATAC and Native-ChIP samples, dissociated cells were washed once with sort media and then resuspended in sort media supplemented with 100 U/mL DNase I (Worthington Biochemical), 4mM MgCl2, and 500ng/mL DAPI (Invitrogen). For Crosslinked-ChIP samples, cells were fixed prior to sorting. Dissociated cells were resuspended in PBS + 1% methanol-free formaldehyde (Pierce). Cells were fixed at room temperature for 5 minutes, and then fixation was quenched by adding 1/10th volume of 1.25M glycine. Fixed cells were pelleted (500 rcf, 5 minutes, room temperature), washed once with PBS + 2% FBS, and then resuspended in sort media. All cells were passed through a 40uM cell strainer prior to sorting.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Crosslinked-ChIP: Chromatin from formaldehyde cross-linked cells was sheared on a Covaris S220 Focused-ultrasonicator to a size range of 200-500bp. Approximately 1-2ug of sheared chromatin was used for immunoprecipitation with antibodies, and precipitated DNA was column purified. Native-ChIP: Native chromatin was digested with Micrococal Nuclease to predominantly mono and di-nucleosome sized fragments. Nucleosomal DNA was immunoprecipitated with antibodies and column purified. ATAC: Cells were lysed and then transposed with Nextera Tn5 transposase (Illumina). Transposed DNA was column purified using a Qiagen MinElute PCR cleanup kit. RNA: Cells were lysed in Trizol-LS (ThermoFisher) then RNA was extracted and treated with Turbo-DNase (ThermoFisher).
Crosslinked-ChIP: ChIP-seq Libraries were prepared using a Nugen Ultralow V2 kit. Native-ChIP: ChIP-seq Libraries were prepared using a Nugen Ultralow V2 kit. ATAC: Transposed DNA was amplified using barcoded primers and NEBNext High-Fidelity PCR Master Mix. RNA: RNA-seq libraries were prepared using a TruSeq Stranded Total RNA with Ribo-Zero Gold Set B kit (Illumina RS-1222302).
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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 |
Adapter Sequences were trimmed with cutadapt (v1.9.1) (--minimum-length 18)
Alignment: All samples were aligned to mm10. Crosslinked ChIP-Seq samples were aligned with Bowtie2 (2.2.6) with default parameters. ATAC-Seq and Native ChIP-Seq samples were aligned with Bowtie2 (2.2.6) using -X 1000.
Filtering: Unmapped reads and reads with a mapping quality below 30 were removed with Samtools (1.3.1). For ATACseq, reads mapping to mitochondrial DNA were also removed. For ATAC and ChIP-seq, duplicate reads were marked with Picard and removed with samtools.
For ChIP-Seq and ATAC-Seq, bigWig files were generated using HOMER (4.7) (-fsize 1e20 -fragLength given). ATAC-seq fragments were shifted 4bp upstream (- adjust 4) during bigWig generation. For RNA-Seq, bigWig files were generated with RSeQC (2.6.4) using -t 10000000.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files at base pair resolution. ChIP-Seq and ATAC-Seq files normalized to a library size of 10,000,000 reads. RNA-seq files normalized to a library size of 1,000,000 reads.
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| Submission date |
Jan 12, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Kevin Monahan |
| E-mail(s) |
km1339@dls.rutgers.edu
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| Organization name |
Rutgers University
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| Department |
Molecular Biology and Biochemistry
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| Street address |
604 Allison Rd
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| City |
Piscataway |
| State/province |
NJ |
| ZIP/Postal code |
08854 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE93570 |
Chromatin State and Binding of Lhx2 and Ebf in Olfactory Sensory Neurons |
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| Relations |
| BioSample |
SAMN06226309 |
| SRA |
SRX2487379 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2454916_mOSN_H3K79me3_Mnase-ChIP.bigWig |
341.5 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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