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Sample GSM3489383 Query DataSets for GSM3489383
Status Public on Nov 27, 2018
Title MeCP2_ChIPRx_GSK343_rep2
Sample type SRA
 
Source name Neuroblastoma
Organism Homo sapiens
Characteristics cell line: SH-SY5Y
cell type: Neuroblastoma
spike-in reference: Drosophila melanogaster
spikein_mix_ratio: 3 to1
treatment: GSK343
chip antibody: MeCP2
Treatment protocol SH-SY5Y cells (density of 0.5~0.6 x 10^6 cells/ml) were treated with either DMSO or 5 uM GSK343 (an inhibitor of H3K27me3-specific HMTase) for 72hours.
Growth protocol SH-SY5Y (Korean Cell Line Bank, 22266) were maintained in DMEM/F12 (Gibco, 11320033) supplemented with 10% fetal bovine serum (Welgene, S001-01) and Penicillin/Streptomycin (Lonza, 17-602E) at 5% CO2 in 37°C. Drosophila S2 cells (ATCC, CRL-1963) were cultured in Schneider’s Drosophila Medium (Gibco, 21720024) supplemented with 10% fetal bovine serum (Welgene, S001-01) in 25°C.
Extracted molecule genomic DNA
Extraction protocol The SH-SY5Y cells were washed with 10ml of PBS and cross-linked with 1% formaldehyde for 5minutes. The crosslinking reactions were quenched with 10x Glycine stop solution. The cross-linked SH-SY5Y cells were lysed in lysis buffer (5 mM PIPES, 85 mM KCl, 0.5% IGEPAL CA-630, pH 8.0) using a dounce homogenizer (Kimble-Kontes. 885300-0002) to aid in nuclei release. The released nuclei was pelleted by centrifugation (10min, 5000rpm at 4°C). As a reference exogenous genome, Drosophila S2 cells were cross linked with 1% formaldehyde for 5 minutes and were quenched with 10x Glycine stop solution. The Drosophila S2 cells were then washed 3 times with ice cold PBS. For each ChIP-Rx experiment, SH-SY5Y cells and drosophila S2 cells ratio of 3:1, 12 million crosslinked SH-SY5Y nuclei and 4 million crosslinked S2 cells were combined and sonicated for 15 minutes with 30 second intervals to shear genomic DNA using Bioruptor XL (Diagenode). Each sheared genomic DNA preparation was split into two equal portions and incubated with either H3K27me3 (Diagenode, pAb-069-050), or MeCP2 (Diagenode, pAb-052-050). A magnetic bar was used to pull down antibody corresponding protein-genomic DNA complex. Genomic DNA in the complex was reverse cross-linked by DNase-free Proteinase K and purified.
H3K27me3 ChIPed or input DNA were used to prepare the ChIP-Seq library as described (NEXTflex ChIP-Seq kit, Bio Scientific, NOVA 5143-01 and NEXTflex ChIP-Seq Barcodes-6, NOVA 514120) followed by high throughput sequencing.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
 
Description processed data file: GSK343_MeCP2.ext200_RPM.bw
Data processing The sequences were separately aligned to either human genome(hg19) or the D. melanogaster melanogaster genome(dm3) using Bowtie 2 with the options --sensitive --score-min L,-1.5,-0.3. PCR duplicate are removed for further analysis.
PCR duplicate are removed using samtools rmdup for further analysis.
For quantitative comparisons of H3K27me3 ChIPed reads between the samples, the exogenous genome derived normalization factor (α-x) for each experiment was derived according to the protocol of Orlando et al. As follows: 1 over the number of reads mapping to drosophila (dm3) per million. Then, the uniquely aligned readings for homo sapiens (hg19) were standardized using the normalization factor, α-x, for quantitative comparisons between samples.
For quantitative comparisons of MeCP2 ChIPed reads between the samples, the normalization factor (α) for each experiment was derived as follow: 1 over the number of reads mapping to homo sapiens (hg19) per million. Then, the uniquely aligned readings for homo sapiens (hg19) were standardized using the normalization factor, α, for quantitative comparisons between samples.
Genome_build: hg19
Supplementary_files_format_and_content: The bigwig files were generated from exogenous reference normalized alignment read using the deepTools package
 
Submission date Nov 27, 2018
Last update date Nov 28, 2018
Contact name Wooje Lee
E-mail(s) ntinamu001@gmail.com
Organization name Chosun University
Department Cellular and Molecular Medicine
Street address 309, Pilmun-daero, Dong-gu
City Gwangju
ZIP/Postal code 61452
Country South Korea
 
Platform ID GPL18573
Series (2)
GSE122364 MeCP2 regulates genome-wide gene expression through recognition of H3K27me3 [ChIP_human]
GSE122366 MeCP2 regulates genome-wide gene expression through recognition of H3K27me3
Relations
BioSample SAMN10484608
SRA SRX5064671

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

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