|
| Status |
Public on May 31, 2021 |
| Title |
H3K27Ac_E24NocR1 |
| Sample type |
SRA |
| |
|
| Source name |
H9 cells
|
| Organism |
Homo sapiens |
| Characteristics |
type: embryonic stem cells
treatment: Mesendoderm 24h + Nocodazole
biological replicate: Replicate 1
|
| Treatment protocol |
H9 cells were synchronized in their cell cycle by incubating them with nocodazole (100 ng/ml) for 16h. Then, nocodazole was withdrawn and cells induced to differentiated into mesendoderm for 24h (In CDM-PVA medium supplemented with Insulin (7ug/ml), Activin A (100 ng/ml), BMP (10 ng/ml), LY294002 (10 mM ), and CHIR99021 (3 mM). To block cells in their S phase during differentiation, we added Hydroxyurea (200 mM) at the start of the differentiation. To block the cells in their G2/M phases during differentiation, we added nocodazole 8h after the induction of the differentiation. In this way, we made sure that the cells were able to re-initiate the cell cycle and get blocked in the following G2/M phase. After 24h of differentiation, cells were collected and processed for ChIP-seq library preparation.
|
| Growth protocol |
H9 cells were grown in E8 medium containing penicillin (50 units/ml) and streptomycin (100 mg/ml)mycin, on plates coated with Vitronectin-XF (Stem Cells Technologies). Cells were incubated at 37ºC in atmosphere of 5% CO2
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were sonicated and histone-DNA complexes were pull-down with antibody.
Libraries were prepared for sequencing using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina; Ref N: E7645S
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
Mesendoderm 24h + Nocodazole
|
| Data processing |
Base calling was done using illumina package RTA v2.7.7, and instrument control package called HCS v3.4.0.38.
Adaptors were clipped before alignment and then re-inserted
Alignment files were converted into BAM files.
Genome_build: GRCh38_genecode
Supplementary_files_format_and_content: All bigWig files were generated using the function bamCompare from the Deeptools software for Histone marks vs Input, with a bin size = 10, normalized using RPKM method, and an effective genome size of 2913022398
|
| |
|
| Submission date |
May 07, 2019 |
| Last update date |
May 31, 2021 |
| Contact name |
Rodrigo Andres Grandy |
| Organization name |
University of Cambridge
|
| Department |
Surgery
|
| Lab |
Vallier Lab
|
| Street address |
Cambridge Stem Cell Institute/ Anne McLaren Laboratory West Forvie Building, Robinson Way
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0SZ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE130826 |
Effect of Cell Division on Cell Differentiation (ChIP-Seq HUvsNoc) |
| GSE130830 |
Effect of Cell Division on Cell Differentiation |
|
| Relations |
| BioSample |
SAMN11587015 |
| SRA |
SRX5802416 |
