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Sample GSM4800830 Query DataSets for GSM4800830
Status Public on Nov 18, 2022
Title 2i-GFPplus-combined-C
Sample type SRA
 
Source name mouse embryonic stem cells
Organism Mus musculus
Characteristics culture method: 2i/LIF cultured
strain: E14 UHRF1-GFP
fluorescence sorting: GFP positive population
Treatment protocol Serum/LIF mESCs were transduced with the TRC lentiviral mouse kinase shRNA library
Growth protocol E14 ESCs in the Serum/LIF state were cultured without feeders in serum containing media (DMEM 4500 mg/l glucose, 4 mM L-glutamine, 110 mg/l sodium pyruvate, 15% fetal bovine serum, 1 U/ml penicillin, 1 μg/ml streptomycin, 0.1 mM nonessential amino acids, 50 μM β-mercaptoethanol and 103 U/ml ESGRO LIF). E14 ESCs in the 2i state were cultured in the 2i media containing serum-free N2B27 – [DMEM/F12, Neurobasal, N2,B27], 103 U/ml ESGRO LIF, PD0325901 [1 μm] and CHIR99021 [3 μM].
Extracted molecule genomic DNA
Extraction protocol DNA was extracted using the DNeasy blood and tissue kit (Qiagen) using the standard procedure described in the handbook
For PCR amplification of the library 2 μg of DNA for each sample was used, with the DNA split into four 500 ng reactions. A one-step PCR approach was used with P5 and P7 primers. The P5 and P7 primer were comprised of the P5/P7 flow-cell attachment sequence, the Illumina sequencing primer and the vector binding sequence (primer sequence in shown in Supplementary Table 1.). In addition, to avoid the issue of reduced sequence diversity faced by amplicon libraries, PCR was performed with a mix of P5 primers containing a stagger region of different length.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
 
Description targeted DNA-seq to amplify shRNA sequence
Data processing Library strategy: shRNA screen
Alignment of sequencing data was performed using Bowtie2 under standard alignment parameters.
The count table was then imported into R and differential representation analysis was performed using the package EdgeR and using the exactTest function
Supplementary_files_format_and_content: Excel file containing the count table containing the representation, as counts, for each sequenced shRNA species in each sample and replicate. Primer sequences included in the file SupplementaryTable1.docx available on the series record.
 
Submission date Sep 23, 2020
Last update date Nov 18, 2022
Contact name Gabriella Ficz
E-mail(s) g.ficz@qmul.ac.uk, gabriella.ficz@googlemail.com
Phone 07557774595
Organization name Barts Cancer Institute, Queen Mary University of London
Street address Centre for Haemato-Oncology
City London
State/province Rest of the World
ZIP/Postal code EC1M 6BQ
Country United Kingdom
 
Platform ID GPL19057
Series (1)
GSE158453 Multiple signalling pathways converge on UHRF1 stability and activity
Relations
BioSample SAMN16247299
SRA SRX9178693

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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