|
| Status |
Public on Aug 20, 2024 |
| Title |
UCLA1 primed hESC, ZIC3-/- +sgZIC2, rep2 (RNA-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-/- (done on ZIC3-/- clone 20)
treatment: ZIC3-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 |
polyA RNA |
| Extraction protocol |
Cells were lysed in RNAzol RT reagent and total RNA was purified from cell extracts according to manufacturer's instructions.
mRNA containing polyA tail were enriched using the NEBNext Poly(A) mRNA Magnetic Isolation Module and libraries were prepared using the Swift RNA library kit or NEBNext Ultra II RNA Library Prep Kit according to respective manufacturers' instructions.
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
passage 27+15+1 (collected 5 days post-nucleofection)
|
| Data processing |
Raw reads were aligned against the GRCh38 reference grenome using STAR.
Reads were filtered, sorted and marked for duplicates using samtools.
Features were obtained GENCODE reference annotation file v34 and raw counts were correponding to each feature was calculated using HTSEQ. TPM values were calculated using StringTie.
Differentially expressed genes were calculated using DESeq2. Raw counts were normalised for sequencing depth and library complexity using DESeq2.
Assembly: GRCh38
Supplementary files format and content: Processed data includes TPM values provided as a .txt file
|
| |
|
| 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) |
| GSE270786 |
ZIC2 and ZIC3 promote SWI/SNF recruitment to safeguard progression towards human primed pluripotency [RNA-seq] |
| GSE270787 |
ZIC2 and ZIC3 promote SWI/SNF recruitment to safeguard progression towards human primed pluripotency |
|
| Relations |
| BioSample |
SAMN42045085 |
| SRA |
SRX25057755 |
