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GEO help: Mouse over screen elements for information. |
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Status |
Public on Aug 07, 2024 |
Title |
gRNA sequencing - Early Pluripotent Biol Rep1 |
Sample type |
SRA |
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Source name |
hybrid 129S1/SvImJ, CAST/EiJ
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Organism |
Mus musculus |
Characteristics |
cell line: hybrid 129S1/SvImJ, CAST/EiJ cell type: reprogramming cell cell type: Early Pluripotent
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Treatment protocol |
Mouse recombinant IFNγ (10 ng/ml) (R&D Systems, 485-MI-100) was added to the reprogramming medium from day 0 to day 5.
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Growth protocol |
Mouse embryonic stem cells were incubated at 37 °C with 5% CO2 and cultured on 0.2% gelatin-coated plates in serum/LIF medium: DMEM medium (Thermo Fisher Scientific, 31966021) supplemented with 10% FBS (ES pre-tested, Capricorn, FBS-ES-12A), 1,000 U/ml LIF (ORF Genetics, 01-A1140-0100), 25mM HEPES (Thermo Fisher Scientific, 15630056), 1mM Sodium Pyruvate (Thermo Fisher Scientific, 11360070), 1x MEM NEAA (Thermo Fisher Scientific, 11140050), 50U/ml penicillin/streptomycin (Ibian Tech, P06-07100) and 0.1mM 2-mercaptoethanol (Thermo Fisher Scientific, 31350010). Neural Precursor cells were differentiated from ESCs and cultured in RHBA medium (Takara Bio, Y40001) supplemented with 10 ng/ml EGF (R&D Systems, 236-EG-200), 10 ng/ml bFGF (Thermo Fisher Scientific, 13256029) and ROCK inhibitor 10 µM (Selleck Chemicals, S1049). Cells undergoing reprogramming were grown on top of male irradiated Mouse Embryonic Fibroblasts (iMEFs) on 0.2% gelatin coated plates in iPSC medium: DMEM medium (Thermo Fisher Scientific, 31966021) supplemented with 15% FBS (ES pre-tested, Capricorn, FBS-ES-12A), 25mM HEPES (Thermo Fisher Scientific, 15630056), 1mM Sodium Pyruvate (Thermo Fisher Scientific, 11360070), 1x MEM NEAA (Thermo Fisher Scientific, 11140050), 50U/ml penicillin/streptomycin (Ibian Tech, P06-07100) and 0.1mM 2-mercaptoethanol (Thermo Fisher Scientific, 31350010), supplemented with 1,000 U/ml LIF, 25 mg/ml L-ascorbic acid and 1 mg/ml doxycycline.
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Extracted molecule |
genomic DNA |
Extraction protocol |
Cells were dissociated and sorted based on BFP, SSEA1-APC and X-GFP expression using a BD FACSAria II SORP. Genomic DNA was extracted from cell pellets using the DNeasy Blood and Tissue Kit (Qiagen, 69504). For amplification of the gRNAs and introduction of the Illumina-sequencing adapters, two consecutive PCRs were performed by using the Q5 High-Fidelity DNA Polymerase (NEB, M0491), adding the Illumina adapsters for NGS. Single-end sequencing (1 x 50 bp) on an Illumina HiSeq 2500. Each sample contains two technical replicates
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2500 |
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Data processing |
The gRNA sequencing from the CRISPR KO screening was analyzed with MAGeCK software (Li et al. 2014). The gRNA abundance of each population was determined by taking into account the two biological replicates (with two technical replicates each). The gRNA abundance comparisons were performed pairwise. Assembly: mm10 Supplementary files format and content: xlsx file containing raw counts for each gRNA in each sample
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Submission date |
Jun 30, 2023 |
Last update date |
Aug 07, 2024 |
Contact name |
Mercedes Barrero |
Organization name |
Centre for Genomic Regulation
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Street address |
Dr. Aiguader, 88
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City |
Barcelona |
ZIP/Postal code |
08003 |
Country |
Spain |
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Platform ID |
GPL17021 |
Series (2) |
GSE236244 |
CRISPR Screen: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming |
GSE236247 |
The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming |
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Relations |
BioSample |
SAMN36174861 |
SRA |
SRX20851011 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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