|
| Status |
Public on Nov 18, 2022 |
| Title |
2i-GFPminus-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 negative 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 |
SAMN16247302 |
| SRA |
SRX9178690 |
