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| Status |
Public on Jan 01, 2024 |
| Title |
NRSF/REST_ChIPseq_Eq_R2 |
| Sample type |
SRA |
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| Source name |
Skin fibroblasts
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| Organism |
Equus caballus |
| Characteristics |
cell type: Skin fibroblasts
passages: 10
strain: E.Derm (NBL-6)-quarterhorse
chip antibody: NRSF monoclonal antibody (Caltech Protein Expression Center, Pasadena, CA, USA, 12C11-1B11)
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| Growth protocol |
Mouse macrophage cells (RAW264.7, ATCC, Manassas, VA) were grown in DMEM medium (30-2002, ATCC, Manassas, VA) supplemented with 10% FBS (VWR, Radnor, PA) and penicillin/streptomycin (Life Technologies). Dog myelomonocytic leukemia cells (ML2, ATCC, Manassas, VA) were grown in RPMI-1640 medium (Life Technologies, Carlsbad, CA) supplemented with 1000 mM Hepes, 10mM MEM NEAA, 100mM Sodium pyruvate, 55mM β- mercaptoethanol, 10% FBS and penicillin/streptomycin. Horse skin fibroblast (E.Derm, ATCC, Manassas, VA) were grown in EMEM(30-2003, ATCC, Manassas, VA) supplemented with 10% FBS and penicillin/streptomycin. Trypan Blue (VWR) staining was performed before harvesting the cells for each assay to make sure that at least 90% of the cells were viable.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
We cross-linked about 20 million cells with 1% formaldehyde for each replicate. Cross-linked chromatin was sonicated into 200-300bp fragments. DNA fragments were incubated with Dynabeads protein G (Thermo Fisher Scientific) bound by NRSF monoclonal antibody (Caltech Protein Expression Center, Pasadena, CA, USA, 12C11-1B11) overnight to perform immunoprecipitation. Then chromatin was reverse cross-linked to extract NRSF-bound DNA.
DNA fragments recovered from ChIPs were end-repaired, ligated to adapters, size selected (200 to 300bp) and PCR-amplified to make the libraries for sequencing. The libraries were made based on Myers Lab protocol v042211 (http://myers.hudsonalpha.org/documents/Myers%20Lab%20ChIP-seq%20Protocol%20v042211.pdf).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
NRSF/REST.peaks.Eq.txt
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| Data processing |
ChIP and input sequencing reads were aligned using Bowtie v.0.12.8
After alignment, peaks were detected using MACS v.1.4.2, retaining all statistically enriched regions ( p<10-4.5). Binding regions were considered reproducible when they were identified in both replicates. Consensus binding regions were then merged and called subpeaks using PeakSplitter_Cpp v.1.0. Subpeaks in 50bp were merged. Peaks having (1) higher signal in control than ChIP replicates in each species or (2) signal in control is higher than 1.4 RPM in each species or (3) in Blacklist (ENCODE Project Consortium. 2012) in human and mouse were filtered out as artificially high signal peaks.
Genome_build: GRCh37/hg19,GRCm38/mm10, CanFam3.1, EquCab2.0
Supplementary_files_format_and_content: peak calling results for each species in .txt format. (chromosome, start, end)
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| Submission date |
Feb 03, 2016 |
| Last update date |
Jan 01, 2024 |
| Contact name |
Shan Jiang |
| E-mail(s) |
jiangs2@uci.edu
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| Organization name |
UNIVERSITY OF CALIFORNIA, IRVINE
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| Lab |
Mortazavi Lab
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| Street address |
2300E Biological Sciences III
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| City |
IRVINE |
| ZIP/Postal code |
92697 |
| Country |
USA |
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| Platform ID |
GPL21401 |
| Series (1) |
| GSE77543 |
Dynamics of NRSF/REST motif evolution favor the canonical NRSE/RE1 form |
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| Relations |
| BioSample |
SAMN04455243 |
| SRA |
SRX1559585 |
| 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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