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Sample GSM2054210 Query DataSets for GSM2054210
Status Public on Jan 01, 2024
Title NRSF/REST_ChIPseq_Cf_R1
Sample type SRA
Source name Myelomonocytic leukemia cells
Organism Canis lupus familiaris
Characteristics cell type: Myelomonocytic leukemia cells
passages: 13-15
strain: ML2
chip antibody: NRSF monoclonal antibody (Caltech Protein Expression Center, Pasadena, CA, USA, 12C11-1B11)
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.
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 (
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
Description NRSF/REST.peaks.Cf.txt
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)
Submission date Feb 03, 2016
Last update date Jan 01, 2024
Contact name Shan Jiang
Lab Mortazavi Lab
Street address 2300E Biological Sciences III
ZIP/Postal code 92697
Country USA
Platform ID GPL21400
Series (1)
GSE77543 Dynamics of NRSF/REST motif evolution favor the canonical NRSE/RE1 form
BioSample SAMN04455239
SRA SRX1559581

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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