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Status |
Public on Sep 22, 2017 |
Title |
mOSN_H3K9me3_Mnase-ChIP_rep2 |
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-H3K9me3 (abcam, ab8898) 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 |
SAMN06226312 |
SRA |
SRX2487376 |
Supplementary file |
Size |
Download |
File type/resource |
GSM2454913_mOSN_H3K9me3_Mnase-ChIP_rep2.bigWig |
505.1 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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