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Status |
Public on Jun 07, 2021 |
Title |
H3K9me2 - Day 0 - GATA6 WT |
Sample type |
SRA |
|
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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)
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Treatment protocol |
Doxycycline was supplemented to induce GATA6-3xFLAG cDNA expression in the denoted samples
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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).
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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.
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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
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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. |
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Relations |
BioSample |
SAMN15784704 |
SRA |
SRX8923842 |