 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 20, 2024 |
| Title |
UCLA1 primed hESC, NT control, H3K27me3 ChIP-seq, rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Primed human embryonic stem cells
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Primed human embryonic stem cells
cell line: UCLA1 primed hESCs
cell type: Human embryonic stem cells
genotype: WT
treatment: none
antibody: anti-H3K27me3 (CST 9733)
passage description: passage 27+9
|
| Treatment protocol |
Cells were treated with 100 ng/ml doxycycline hyclate, 1 uM flavopiridol, 500 nM triptolide, 25 uM STM2457 or DMSO of equivalent v/v concentration, where specified for the appropriate period of time.
|
| Growth protocol |
Primed hESCs were cultured on hESC-qualified matrigel coated plates in TESR-E8 or mTESR-plus medium. Primed hESCs were cultured in media supplemented with 10 µM Y27632 24h prior to eletroporation experiments and upon plating of electroporated cells. hESCs collected during reversion from primed to naive pluripotency were plated on a feeder layer of mouse embryonic fibroblasts and cultured in 5iLAF or 4iLAF+0.25µM IM-12 (t5iLAF) medium for the specified period of time. Both during reversion to and upon full acquisition of naive pluripotency, hESCs were cultured in 5 % O2 and 5 % CO2. Reverting and naive hESCs were depleted of MEFs by plating of dissociated single cell suspension on 0.1% gelatin for 30 minutes prior to collection for experiments.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% paraformaldehyde for 10 min, and quenched with 0.14 M glycine for 10 min. Cells were then resuspended with 1 ml lysis buffer 1 (10 mM TrisHCl pH 8.0, 0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 1x Protease inhibitors (Roche), 1 mM NaF, 1 mM Na2VO3 and 1 mM PMSF), then rotated 15 minutes. Nuclei were pelleted by centrifugation at 1200g for 5 minutes at 4 °C. Nuclei were then resuspended with 1 ml lysis buffer 2 (10 mM TrisHCl pH 8.0, 200 mM NaCl, 10 mM EDTA, 0.5 mM EGTA, 1x Protease inhibitor, 1mM NaF, 1mM Na2VO3 and 1mM PMSF) and rotated 10 minutes at 4 °C. Nuclei were then pelleted and resuspended in 900 μl lysis buffer 3 (10 mM Tris-HCL pH 8, 10 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 1 mM NaF, 1 mM Na2VO3 and 1 mM PMSF), and chromatin was fragmented using the Covaris M220 sonicator. Chromatin was pre-cleared with ProtA/G beads for 2 hours at 4 °C. Antibodies were then added to pre-cleared lysates and incubated overnight with end-over-end shaking at 4 °C. Immunocomplexes were then immobiilzed on prot A/G beads, which were then washed 2×4 minutes with 500 μl Wash Buffer A (50 mM HEPES pH 7.9, 1% TritonX-100, 0.1% Deoxycholate, 1mM EDTA, 140 mM NaCl), 500 μl Wash Buffer B (50 mM HEPES pH 7.9, 0.1% SDS, 1% Triton X100, 0.1% Deoxycholate, 1 mM EDTA, 500 mM NaCl), and 500 μl TE buffer (10 mM TrisHCl pH 8.0, 1 mM EDTA). DNA was eluted in 100 μl elution buffer (50 mM TrisHCl pH 8.0, 1 mM EDTA, 1% SDS) after incubation at 65 °C for 10 minutes at 1400 rpm. The elute was collected, and the beads were subjected to a second round of elution with 150 μl elution buffer. The ChIP eluants were pooled, and the input sample was diluted to 250 μl with elution buffer. These samples were incubated 65 °C overnight to promote decrosslinking. The samples were then allowed to cool to room temperature, 15 μg of RNAse A (Purelink, ThermoFisher) was added, and the samples were incubated 30 minutes at 37 °C to degrade RNA. 100 μg Proteinase K was then added and the samples were incubated 56 °C for two hours. DNA was purified using a MinElute PCR Purification kit (Qiagen).
Purified DNA was fragmented to 200 bp by sonication. Following this, libraires were prepared using the Swift DNA library kit or NEBNext Ultra II DNA Library Prep Kit, according to manufacturers' instructions.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
Reads were aligned against the GRCh38 reference genome using bwa mem.
Reads were filtered for unique alignments, marked for duplicates and sorted using samtools
Effciency biases were calculated using csaw and scale factor was calculated to make coverage tracks, where necessary. Otherwise reads per 1x genome coverage (RPGC) was used for generating coverage files.
Differentially accessible regions were calculated using csaw.
Deeptools was used to generate coverage tracks.
Assembly: GRCh38
Supplementary files format and content: bigWig
|
| |
|
| Submission date |
Jun 25, 2024 |
| Last update date |
Aug 20, 2024 |
| Contact name |
William A. Pastor |
| E-mail(s) |
william.pastor@mcgill.ca
|
| Phone |
5143988962
|
| Organization name |
McGill University
|
| Department |
Biochemistry
|
| Street address |
3655 Promenade Sir-William-Osler
|
| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H3G1Y6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL20795 |
| Series (2) |
| GSE270784 |
ZIC2 and ZIC3 promote SWI/SNF recruitment to safeguard progression towards human primed pluripotency [ChIP-seq] |
| GSE270787 |
ZIC2 and ZIC3 promote SWI/SNF recruitment to safeguard progression towards human primed pluripotency |
|
| Relations |
| BioSample |
SAMN42045015 |
| SRA |
SRX25058332 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8351893_Primed_ZIC2WT2_newUCLA1_H3K27me3_ChIP.final.norm.bw |
58.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
