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| Status |
Public on Dec 01, 2020 |
| Title |
MKG108_OVCAR8 AZ_300:3 |
| Sample type |
SRA |
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| Source name |
OVCAR8 AZ_300:3
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| Organism |
Homo sapiens |
| Characteristics |
cell line background: OVCAR8
cell type: high grade serous ovarian cancer cell line
clone phentoype/genotype: C3A1; OVCAR8-derived clones resistant to 300nM AZD1775
treatment: AZD1775
concentration (nm): 300
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| Treatment protocol |
To isolate clones resistant to Wee1 inhibitor AZD1775 (S1525, Selleckchem, Houston, TX), cells were plated at 500 and 1000 cells/well in 96-well plates in medium containing the selective AZD1775 concentration. Single colonies arising in individual wells were picked and expanded for analysis under continuous selection. 16 samples (MKG1-16) from ES-2 and ES-2 derived resistant clones: duplicate cultures of untreated ES-2 parent and ES-2 parent treated for 24 hours with 100nM AZD1775, triplicate cultures of four resistant clones maintained in AZD1775- two clones resistant to 1000nM AZD1775 and their progenitors resistant to 250nM AZD1775.
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| Growth protocol |
All experiments were performed on cultures within 10 passages of supply. Cell lines were cultured in RPMI-1640 medium (OVCAR8; 21875, Thermo Fisher Scientific), supplemented with 10% FCS, Non-Essential Amino Acids (11140-035, Thermo Fisher Scientific), 10% FCS, Non-Essential Amino Acids (11140-035, Thermo Fisher Scientific), 1 mM Sodium Pyruvate, 2 mM L-Glutamine and Penicillin (100 U/ml) - Streptomycin (100 mg/ml) at 37˚C, 5% CO2 mM Sodium Pyruvate, 2 mM L-Glutamine and Penicillin (100 U/ml) - Streptomycin (100 mg/ml) at 37˚C, 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were grown in 25cm2 flasks until they reached 70% confluency then total RNA was extracted using the RNeasy mini kit (74104, Qiagen Ltd., Manchester, UK) according to the manufacturers’ instructions.
Libraries were prepared from each total-RNA sample using the TruSeq Stranded Total RNA with Ribo-Zero Gold kit (#RS-122-2302) according to the provided protocol. 500ng of total-RNA was processed to deplete rRNA before being purified, fragmented and primed with random hexamers. Primed RNA fragments were reverse transcribed into first strand cDNA using reverse transcriptase and random primers. RNA templates were removed and a replacement strand synthesised incorporating dUTP in place of dTTP to generate ds cDNA. AMPure XP beads (Beckman Coulter, #A63881) were then used to separate the ds cDNA from the second strand reaction mix, providing blunt-ended cDNA. A single 'A' nucleotide was added to the 3' ends of the blunt fragments to prevent them from ligating to another during the subsequent adapter ligation reaction, and a corresponding single 'T' nucleotide on the 3' end of the adapter provided a complementary overhang for ligating the adapter to the fragment. Multiple indexing adapters were then ligated to the ends of the ds cDNA to prepare them for hybridisation onto a flow cell, before 12 cycles of PCR were used to selectively enrich those DNA fragments that had adapter molecules on both ends and amplify the amount of DNA in the library suitable for sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
MKG-108_S10
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| Data processing |
Salmon index was created uinsg salmon (v0.10.2) index options -k 31
Read quantitiation was performed using salmon (v0.10.2) quant using options -l ISR --gcBias
Genome_build: hg38
Supplementary_files_format_and_content: tab delmited files contain read counts and TPM values per transcripts
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| Submission date |
Dec 13, 2019 |
| Last update date |
Dec 02, 2020 |
| Contact name |
Graeme Richard Grimes |
| E-mail(s) |
[email protected]
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| Organization name |
University of Edinburgh, The Institute of Genetics and Cancer
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| Department |
Human Genetics Unit
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| Lab |
Bioinformatics Analysis Core
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| Street address |
Western General Hospital, Crewe Road
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| City |
Edinburgh |
| ZIP/Postal code |
EH4 2XU |
| Country |
United Kingdom |
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| Platform ID |
GPL21697 |
| Series (2) |
| GSE141990 |
Identifying and overcoming a mechanism of resistance to Wee1 kinase inhibitor AZD1775 in high grade serous ovarian cancer cells (OVCAR8) |
| GSE142126 |
Identifying and overcoming a mechanism of resistance to Wee1 kinase inhibitor AZD1775 in high grade serous ovarian cancer cells |
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| Relations |
| BioSample |
SAMN13558650 |
| SRA |
SRX7366854 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4217437_MKG108_quant.sf.txt.gz |
2.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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