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Sample GSM4720528 Query DataSets for GSM4720528
Status Public on Jun 07, 2021
Title H3K9me2 - Day 0 - GATA6 WT
Sample type SRA
 
Source name K3_GATA6+/+
Organism Homo sapiens
Characteristics cell type: K3 Induced pluripotent stem cell
genotype: GATA6+/+
differentiation day: Day 0 - Pluripotent stem cells
doxycyline treatment: untreated
chip antibody: H3K9me2 (Abcam; ab1220)
Treatment protocol Doxycycline was supplemented to induce GATA6-3xFLAG cDNA expression in the denoted samples
Growth protocol Pluripotent stem cells were seeded on Geltrex-coated tissue culture plates. After 24 hours, the cells were subjected to hepatic differentiation. Briefly, iPSCs were supplemented with Activin A (100ng/ml), fibroblast growth factor 2 (FGF2 20ng/ml), and bone morphogenetic protein 4 (BMP4, 10ng/ml) for 2 days. Activin A (100ng/ml) alone was supplemented for a further 3 days to form definitive endoderm. Subsequently, the definitive endoderm was specified towards a hepatic fate by supplementing the media with 3 days of BMP4 (20ng/ml) and FGF2 (10ng/ml).
Extracted molecule genomic DNA
Extraction protocol Cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature. The cross-linking reaction was quenched by adding 125mM glycine and incubating at room temperature for 5 minutes. Chromatin was isolated from cell pellets using MNase enzymatic digestion following the manufacturers protocol (Magnetic CHIP kit, ThermoFisher/Pierce). Briefly, cell nuclei were isolated using a membrane lysis buffer. MNase was then added to the isolated nuclei at optimized concentrations and incubated at 37°C for 15 minutes. The enzymatic reaction was stopped with EDTA and digested chromatin was then extracted by brief sonication. Chromatin isolated from 4 individual differentiations were pooled for each condition. Pooled chromatin was incubated with primary antibodies overnight at 4°C. The following day, protein A/G magnetic beads were added to the sample-antibody mixture and incubated at 4°C for 2 hours. Beads were then washed three times in a low salt wash buffer, and once in a high salt wash buffer, before reverse cross-linking in elution buffer. Eluted samples were subjected to proteinase K digestion and the DNA isolated using DNA-binding columns.
CHIP DNA samples were submitted to Beijing Genomics Institute (BGI), who performed library preparation and sequencing on the BGISEQ-500 platform. 50bp single end reads were generated and provided as FASTQ files.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model BGISEQ-500
 
Data processing FASTQC (v0.11.7) of raw sequencing file
Aligned reads to hg19 genome using Bowtie 2 (v.2.2.5)
Removed unaligned reads with Partek Flow software (v.9.0.20.0526)
bedGraph generation performed using HOMER software (v.4.10)
Genome_build: hg19
Supplementary_files_format_and_content: bedGraph files generated using the makeUCSCfile.pl script in HOMER using default settings
 
Submission date Aug 11, 2020
Last update date Jun 07, 2021
Contact name Stephen A Duncan
E-mail(s) [email protected]
Phone 843-792-9104
Organization name Medical University South Carolina
Department Regenerative Medicine and Cell Biology
Lab Duncan Lab
Street address 173 Ashley Avenue, BSB 6th Floor, Room 657A
City Charleston
State/province SC
ZIP/Postal code 29425
Country USA
 
Platform ID GPL23227
Series (2)
GSE156019 GATA6 defines endoderm fate by controlling chromatin accessibility during differentiation of human induced pluripotent stem cells [ChIP-seq]
GSE156021 GATA6 defines endoderm fate by controlling chromatin accessibility during differentiation of human induced pluripotent stem cells.
Relations
BioSample SAMN15784704
SRA SRX8923842

Supplementary file Size Download File type/resource
GSM4720528_H3K9me2_WT_Day_0.ucsc.bedGraph.gz 170.8 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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