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Sample GSM2386579 Query DataSets for GSM2386579
Status Public on Apr 17, 2017
Title M3_2
Sample type SRA
 
Source name H1 POU5F1-IRES-eGFP
Organism Homo sapiens
Characteristics cell type: H1 POU5F4-eGFP reporter cells
Treatment protocol Regular hESC culture without any special treatment.
The core promoter of MSH5 and PRRC2A was deleted by a pair of CRISPR/Cas9 targeting- 150bp to +100bp region near TSS. After picking single clones and Sanger sequencing verification of the genotype, the mutant cells and wild-type control cells were subjected to ATAC-seq analysis.
Growth protocol Cells were cultured at 37 celsius degree in 5% CO2, in Essential 8 media (Stemcell Technologies).
Extracted molecule genomic DNA
Extraction protocol ATAC-seq was performed by following the protocol described earlier (Buenrostro et al, Nature Method, 2013).
Briefly, each library starts with 100k cells which were permeabilized with NPB (0.2% NP-40, 5%BSA, 1Mm DTT in PBS with one complete proteinase inhibitor) at 4 degree for 10min, followed by spin down at 500g for 5min. The resulting nuclei were resuspended in 20ul 1xDMF (33mM Tris-acetate (pH=7.8), 166mM K-Acetate, 10mM Mg-Acetate, 16 % DMF). The chromatin tagmentation was done by adding 0.5ul Tn5 into 10ul solution for 30min at 37 degrees, followed by standar Illumina Tru-seq library prep protocol.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 4000
 
Description ATAC-seq using WT and mutant POU5F1-eGFP reporter hESC.
Mutant clones with MSH5 core promoter deletion derived from POU5F1-eGFP H1 hESC reporter cells purchased from WiWell Institute.
Data processing Library strategy: ATAC-seq
We processed our ATAC-seq data in the following steps: 1) ATAC-seq sequencing reads were mapped to hg19 reference genome using Bowtie(61) in pair-end mode; 2) poorly mapped, improperly paired and mitochondrial reads were filtered; 3) PCR duplications were further removed using Picards MarkDuplicates (http://broadinstitute.github.io/picard.); 4) Mapping positions of reads were adjusted accounting for Tn5 insertion; 6) Reads were next shifted for 75bp followed by peak calling using MACS2(71) with following parameters “-q 0.01 --nomodel --shift 175 –B --SPMR --keep-dup all --call-summits”; 7) ATAC-seq signal was normalized into RPKM using deeptools(72) for visualization.
Genome_build: hg19
Supplementary_files_format_and_content: .bw
 
Submission date Nov 09, 2016
Last update date May 15, 2019
Contact name Bing Ren
Organization name University of California, San Diego School of Medicine
Street address 9500 Gilman Dr., CMM-East, Admin Area/Rm 2071
City La Jolla
State/province CA
ZIP/Postal code 92093
Country USA
 
Platform ID GPL20301
Series (1)
GSE81026 Cis Regulatory Element Scan by Tiling-deletion and Sequencing
Relations
BioSample SAMN06006851
SRA SRX2338899

Supplementary file Size Download File type/resource
GSM2386579_M3_2.bw 209.6 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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