|
| Status |
Public on Jul 08, 2020 |
| Title |
REM_RR_2h_2g |
| Sample type |
SRA |
| |
|
| Source name |
Dog breast cancer cell line model
|
| Organism |
Canis lupus familiaris |
| Characteristics |
cell type: Canine breast cancer
parental cell line: REM
timepoint: 2h post-treatment
treatment: 2 Gray radiation dose
|
| Treatment protocol |
Cells were irradiated using a Faxitron cabinet X-ray system 43855D. Radioresistant cells (REM-RR) were developed from their parental cell line (REM) 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.
Prepared using the standard protocol for the 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 |
REMData_Raw.txt, REMData_Processed.txt, ProcessedData_Combined_QN.txt
|
| 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.
Further filtering performed to select only genes that mapped across species.
All preprocessed data was combined and quantile normalisation was performed using R the preprocessorCore package by Bioconductor.
Where data had 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, filtering and quantile normalisation as decribed.
Supplementary_files_format_and_content: REMData_Raw.txt: Original REM unfiltered data containing raw counts – gene IDs are ENSCAFG from the Canis genome
Supplementary_files_format_and_content: REMData_Processed.txt: Processed REM data after log2 transformation and filtering – to include only genes expressed across all samples and only genes which mapped to ENSM (human IDs).
Supplementary_files_format_and_content: ProcessedData_Combined_QN.txt: Processed data from all samples (canine and human breast cancer cell lines) combined and quantile-normalised.
|
| |
|
| Submission date |
May 06, 2020 |
| Last update date |
Jul 08, 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 |
GPL28490 |
| Series (1) |
| GSE149988 |
Comparative Analysis of the Development of Acquired Radioresistance in Canine and Human Mammary Cancer Cell Lines |
|
| Relations |
| BioSample |
SAMN14843937 |
| SRA |
SRX8272849 |
