NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM1827604 Query DataSets for GSM1827604
Status Public on Mar 24, 2016
Title MeCP2_ChIP_WT_rep1
Sample type SRA
 
Source name MeCP2_ChIP_WT
Organism Mus musculus
Characteristics strain background: B6.129P2(C)-Mecp2tm1.1Bird/J X C57BL/6J
genotype/variation: wild-type, MeCP2(+/y)
age: 8 weeks
tissue: Olfactory epithelium
chip antibody: MeCP2 antibody
chip antibody vendor: Diagenode
chip antibody cat. #: pAb-052-050
Growth protocol Animal care and experimental procedures were conducted in accordance with institution guidelines and complied with the National Institute of Health (NIH) policy. To ensure minimum and equal olfactory stimulation, experimental animals are individually housed in circulating clean air cages for minimum of 24 hours before use. Male Mecp2-/y (KO) and Mecp2+/y (WT) littermates were obtained by crossing heterozygous Mecp2-/+ female (Jackson Laboratory strain: B6.129P2(C)-Mecp2tm1.1Bird/J, stock number 003890) (Guy et al. 2001) with inbred C57BL/6J male.
Extracted molecule genomic DNA
Extraction protocol ChIP-seq was done based on methods established previously (Li, Y et al. 2014). Two independent chromatin immunoprecipitation assays were performed using MAGnify chromatin immunoprecipitation kit (Life Technologies, Grand Island, NY). Specifically, main olfactory neuroepithelia (MOE) were dissected from the nasal cavity and dissociated mechanically via trituration in phosphate buffer saline (PBS). Equal number of cells was used for subsequent steps. Protein-DNA complexes were cross linked by incubating dissociated MOE cells with 1% formaldehyde for 5 minutes on a rocker at room temperature. The cross-linking was quenched by adding Glycine. After washing off the media, MOE cells were lysed briefly in SDS containing buffer followed by sonication for 15 minutes with 30 second intervals to shear genomic DNA using a BioruptorTM 300 (Diagenode, Denville, NJ). Sheared DNA was evaluated by 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and selected for sizes ranging between 200 bp and 300 bp. Each sheared genomic DNA preparation was split into two equal portions and incubated with MeCP2 antibody (Diagenode, pAb-052-050) or Rabbit IgG (Millipore, Cat# 12-370) for the negative control. MeCP2-genomic DNA complexes were pulled down and reverse cross-linked by DNase-free Proteinase K and subsequently purified. MeCP2 ChIP-seq and input DNA library were prepared according to manufacturer’s instruction (Bioo Scientific, Austin, TX) using 5143-01 NEXTflex ChIP-Seq kit and 514120 NEXTflex ChIP-Seq Barcodes-6. ChIP and Input DNAs were PCR amplified, cleaned up and sequenced on Illumina Hi-Seq 2000 at QB3 Vincent J. Coates Genomics Sequencing Laboratory in the UC Berkeley.
library construction protocol
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
 
Data processing ChIP-Seq: The MeCP2 ChIP-seq and Input reads were aligned to the mm9 reference genome using Bowtie 2 using the options --sensitive --score-min L,-1.5,-0.3. Reads mapping to multiple sites in the genome were discarded. PCR-duplicates were removed by keeping at most one mapped read at each position in the genome. The fragment coverages of the ChIP and Input sets were calculated after extending the map coordinate by 200 bp (the experimentally determined fragment length).
MNase-Seq:The MNase-seq reads were aligned to the mm9 reference genome using Bowtie 2 using the options --sensitive --score-min L,-1.5,-0.3. Reads mapping to multiple sites in the genome were discarded. The nucleosome occupancy was calculated by extending the map coordinates 146 bp and tabulating the coverage.
RNA-Seq: The RNA-seq reads were aligned to the mm9 refGene using TopHat 2. Only reads mapping uniquely, in proper pairs, and to the same chromosome were retained, leaving 65M, 55M and 56M reads in the WT replicates and 58M, 51M and 51M reads in the KO replicates. Differential gene expression and statistical significance were calculated for the refGene transcripts using cufflinks and cuffdiff with default parameters but keeping only uniquely mapped and proper read pairs.
Bisulfite-Seq: Deduplication of the raw sequencing reads resulted in 161M and 152M reads constituting combined 21x genomic coverage. We aligned the deduplicated reads to the mm9 reference assembly using Bismark (Krueger et al. 2011) package with Bowtie2 aligner, using default Bowtie2 alignment scoring. Methylation calls were performed by Bismark package.
Genome_build: mm9
Supplementary_files_format_and_content: bigWig files were generated using bedTools genomeCoverageBed and UCSC bedGraphToBigWig. The genes.fpkm_tracking were generated using cufflinks. The Bisulfite-Seq tables were generated usin Bismark.
 
Submission date Jul 20, 2015
Last update date May 15, 2019
Contact name Hans Tomas Rube
Organization name University of Illinois at Urbana Champaign
Department Physics
Lab Jun Song
Street address 1110 West Green Street
City Urbana
State/province Illinois
ZIP/Postal code 61801
Country USA
 
Platform ID GPL13112
Series (1)
GSE71126 Sequence Features Accurately Predict Genome-wide MeCP2 Binding in vivo
Relations
Reanalyzed by GSM3577864
BioSample SAMN03890972
SRA SRX1113143

Supplementary file Size Download File type/resource
GSM1827604_MeCP2_ChIP_WT_rep1.extend200.coverage.mm9.bw 496.1 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap