 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 31, 2021 |
| Title |
R-1095_UWB1.289_40uM_CT_M1 |
| Sample type |
SRA |
| |
|
| Source name |
ovarian cancer cell line UWB1.289
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: UWB1.289
treatment: 40uM CT913-M1
|
| Treatment protocol |
For Whole-Transcriptome Sequencing (RNA-Seq), 3.5x10E6 cells (UWB1.289) or 3.3x10E6 cells (UWB1.289+BRCA1) were seeded in T-75 flasks. After 24h, medium was removed and medium with 1000 µM CT913 and 40µM CT913-M1 or standard growth medium (as control) was added.
|
| Growth protocol |
UWB1.289 ovarian cancer cells: 1:1 mixture of RPMI-1640 and MEGM Bullet Kit, 10 % fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin; UWB1.289+BRCA1 ovarian cancer cells: 50% RPMI-1640 and 50% MEGM Bullet Kit, 10 % fetal calf serum, 100 U/ml penicillin, 100 mg/ml, streptomycin and 200 µg/ml Geneticin/G-418
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA extraction was performed according to the miRNeasy Mini Kit (Qiagen, Hilden, Germany). On column DNA digestion was included into this protocol, in order to remove residual contaminating genomic DNA. All experiments were done according to the manufacturer’s instructions.
For library preparation we used the TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer’s protocol, starting with 1 µg total RNA. All barcoded libraries were pooled and sequenced 2x75bp paired-end on an Illumina NextSeq500 platform to obtain a minimum of 10 Mio reads per sample.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
Raw reads were converted from bcl to fastq format using bcl2fastq (version v2.17.1.14) allowing for 1 barcode mismatch
Reads were trimmed for quality, sequence adapters and cropped to 75nt using trimmomatic 0.36 with the following parameters: ILLUMINACLIP:TruSeq3-PE.fa:2:30:10:2:true LEADING:15 TRAILING:15 SLIDINGWINDOW:4:15 MINLEN:36 CROP:75
Reads were aligned against the genome STAR 2.5.1 in a 2-pass mapping mode: first, an index was created using the genome sequence and gene annotation (here, Gencode GRCh38.p7 comprehensive gene annotation), against which all reads were aligned. Second, all detected splice junctions of all samples were merged and used as guide for the second mapping step. The following parameters were used in both steps: --readFilesCommand zcat --alignIntronMax 500000 --alignMatesGapMax 500000 --outSAMtype BAM SortedByCoordinate --outSAMprimaryFlag OneBestScore --outFilterMultimapNmax 100 --outFilterMismatchNoverLmax 0.05 --chimSegmentMin 15 --chimScoreMin 1 --chimScoreJunctionNonGTAG 0 --chimJunctionOverhangMin 15 --chimSegmentReadGapMax 3 --alignSJstitchMismatchNmax 5 -1 5 5. For the second step, additional parameters are: --limitSjdbInsertNsj 10000000 --limitBAMsortRAM 20000000000 --limitGenomeGenerateRAM 20000000000 --sjdbFileChrStartEnd allSJ.out.tab, where allSJ. out.tab denotes the collected splice junctions.
Read counts of all annotated genes were extracted from the alignments using the featureCounts function of the Rsubread package (v1.20.6) with the following parameters: GTF.featureType = “exon”, GTF.attrType=“gene_id”, useMetaFeatures = T, isPairedEnd=T, requireBothEndsMapped=F, allowMultiOverlap=T, countMultiMappingReads=F, fraction=T. Counts of rRNA, and genes with 0 counts for all samples were discarded
Expression was measured as regularized-logarithm transformation (DESeq2, v1.10.1) of transcripts per kilobase million (TPM)
Genome_build: hs37d5 (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz)
Supplementary_files_format_and_content: read counts from data processing steps above (before DESeq application), columns represent samples, rows represent genes (Ensembl Gene ID)
|
| |
|
| Submission date |
May 12, 2020 |
| Last update date |
May 31, 2021 |
| Contact name |
Konrad Grützmann |
| E-mail(s) |
konrad.gruetzmann@uniklinikum-dresden.de
|
| Phone |
+49 351 458 19621
|
| Organization name |
National Center for Tumor Diseases partner site Dresden
|
| Department |
Core Unit for Molecular Tumor Diagnostics
|
| Street address |
Schubertstraße 15
|
| City |
Dresden |
| ZIP/Postal code |
01307 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21697 |
| Series (1) |
| GSE150377 |
Transcriptomic analysis (RNA-seq) of BRCA1-proficient and BRCA1-deficient ovarian cancer cells treated with the triazene compound CT913 and its metabolite CT913-M1 |
|
| Relations |
| BioSample |
SAMN14896883 |
| SRA |
SRX8330125 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
