|
| Status |
Public on Aug 20, 2024 |
| Title |
UCLA1 primed hESC, ZIC3-/-ZIC5-/- +sgZIC2, rep1 (ATAC-seq) |
| 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: ZIC2-/-ZIC3-/-ZIC5-/-
treatment: ZIC3- and ZIC5-deficient primed hESCs electroporated with Cas9 and sgRNAs targeting ZIC2
|
| Treatment protocol |
Cells were treated with 100 ng/ml doxycycline hyclate or DMSO of equivalent v/v concentration, where specified.
|
| Growth protocol |
Primed hESCs were cultured on hESC-qualified matrigel coated plates in TESR-E8 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 prior to collection for experiments.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysis in 50 ul ATAC resuspension buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl and 3 mM MgCl2) supplemented with 0.1% Tween-20, 0.1% NP-40 and 0.01% digitonin, and incubated on ice for three minutes. Lysis buffer was then diluted with 1 ml ATAC resuspension buffer supplemented with 0.1% Tween-20 and tubes were inverted three times. Nuclei were pelleted by centrifugation at 500g for 10 minutes at 4°C. Transposition reaction was carried out in 50 ul volume for each sample by resuspending nuclei in 25 ul 2x TD buffer (20 mM Tris-HCl pH 7.6, 10 mM MgCl2 and 20 DMF), 2.5 ul transposase (100nM final), 16.5 ul PBS, 0.5 ul 1% digitonin, 0.5 ul 10% Tween-20 and 5 ul H2O. Reactions were incubated at 37°C for 30 minutes in a thermomixer with 1000 RPM mixing. Reactions were quenched with 5x volume of buffer PB and DNA was purified using the MinElute PCR purification kit (Qiagen). Libraries were next amplified using Illumina-compatible primers.
Libraries were constructed as described in Corces et al (2017) Nature Methods.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
P27+12+7+1 (collected 5 days post-nuclefection)
|
| Data processing |
Raw reads were trimmed to 50bp using TrimGalore!
Reads were aligned against the GRCh38 reference genome using bwa mem.
Reads were filtered for unique alignments, marked for duplicates and sorted using samtools
Mitochondrial reads were removed prior to making coverage files and differential analysis.
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.
Assembly: GRCh38
Supplementary files format and content: bigWig
|
| |
|
| Submission date |
Jun 27, 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) |
| GSE270787 |
ZIC2 and ZIC3 promote SWI/SNF recruitment to safeguard progression towards human primed pluripotency |
| GSE270948 |
ZIC2 and ZIC3 promote SWI/SNF recruitment to safeguard progression towards human primed pluripotency [ATAC-Seq] |
|
| Relations |
| BioSample |
SAMN42143768 |
| SRA |
SRX25140086 |
