|
| Status |
Public on Oct 04, 2018 |
| Title |
ZR_RR_8h_2g_S49 |
| Sample type |
SRA |
| |
|
| Source name |
Breast cancer cell line model
|
| Organism |
Homo sapiens |
| Characteristics |
parental cell line: ZR-751
cell type: Radioresistant breast cancer cell line
time point: 8h post-radiation
|
| Treatment protocol |
Cells were irradiated using a Faxitron cabinet X-ray system 43855D.
Radioresistant cell lines (MCF-7 RR, ZR-751 RR and MDA-MB-231 RR) were developed from their respective parental cell lines (MCF-7, ZR-751 and MDA-MB-231) by weekly exposure to single fractions of radiation. Radiation doses were increased from 0.5 Gy to 10 Gy over a period of 12 weeks and then maintained by weekly doses.
|
| Growth protocol |
All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum (FCS), 50 U ml-1 penicillin and 50 mg ml-1 streptomycin and incubated at 37oC in a humidified atmosphere with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Pellets of up to 1x10^7 cells were collected by trypsinization at 0, 2 and 8 h post-radiation, snap-frozen on dry ice and stored at – 70oC for subsequent RNA extraction. Total RNA was extracted from cells with the RNeasy Mini Kit using QIAshredder technology (UK Qiagen, Ltd). The manufacturer’s protocol for purification of total RNA from animal cells using spin technology was followed.
Lexogen QuantSeq 3' mRNA-Seq Prep Kit for Illumina (FWD)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
ZR RR 8h 2g
|
| Data processing |
Total RNA samples underwent quality control using Agilent Bioanalyser and RNA 600 Nanochips and quantified using the Qubit 2.0 fluorometer and the RNA BR assay.
Libraries generated from 500ng of each total RNA sample using the Lexogen QuantSeq 3' mRNA-Seq Library Prep Kit for Illumina (FWD).
After amplification and purification, libraries were quantified using the Qubit dsDNA HS assay kit and assessed for size/quality using Agilent DNA HS kit.
Sequencing performed using the NextSeq 500/550 High-Output v2 kit (#FC-404-2005) on the NextSeq 550 platform (Illumina, #SY-415-1002).
Demultiplexed FASTQ files were generated using Bcl2fastq2 v2.17.1.14 software provided by Illumina.
Individual FASTQ files processed using the BlueBee software provided by Lexogen: trimming of low quality tails and adapter contamination, alighment with ENCODE settings and generate read counts using HTSeq-count.
Working on MS Excel: transfer gene read counts from each sample on to a single file, filter genes with low read counts for the majority of samples, relabel genes to Ensemble IDs obtained from Biomart and do log2 transformation.
Perform quantile normalisation using R the preprocessorCore package by Bioconductor.
Where data that to be integrated with data from publically-available datasets, integration batch effects were corrected using ComBat (Bioconductor).
Genome_build: GRCh38
Supplementary_files_format_and_content: .txt file containing read counts per gene for every samples after filtering, log2 transformation and quantile normalisation as decribed.
|
| |
|
| Submission date |
Oct 03, 2018 |
| Last update date |
May 06, 2020 |
| Contact name |
Carlos Martinez-Perez |
| E-mail(s) |
[email protected]
|
| Organization name |
The University of Edinburgh
|
| Department |
Institute of Genetics and Molecular Medicine
|
| Street address |
Western General Hospital, Crewe Road South
|
| City |
Edinburgh |
| ZIP/Postal code |
EH4 2XU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL21697 |
| Series (1) |
| GSE120798 |
Development and Characterisation of Acquired Radioresistant Breast Cancer Cell Lines. |
|
| Relations |
| Reanalyzed by |
GSE149988 |
| BioSample |
SAMN10172528 |
| SRA |
SRX4795029 |
