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| Status |
Public on May 29, 2019 |
| Title |
48h_2mg_CKI3_Rep3_Batch2 |
| Sample type |
SRA |
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| Source name |
total RNAs, cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MDA-MB-231
tumor type: breast cancer
treatment: CKI
time: 48h
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| Treatment protocol |
Drug treatments were as follows: CKI (2.0 mg/m) or fractionated and reconstituted mixtures (2 mg/ml equivalent) for 24 and 48 hours
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| Growth protocol |
MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC, VA, USA). HEK-293 and HFF were kindly provided by Prof. Andrea Yool (Medical School, University of Adelaide). Cells were cultured in Dulbecco's Modified Eagle's Medium (Thermo Fisher Scientific) with 10 % Fetal bovine serum (Thermo Fisher Scientific) at 370C with 5 % CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by using an RNA extraction kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and RNA samples were quantified and quality determined using a Bioanalyzer at the Cancer Genome Facility of the Australian Cancer Research Foundatin (Australia).
RNA samples with RNA integrity number (RINs) > 7.0 were sent to be sequenced at Novogene (China). Briefly, after QC procedures were performed, mRNA was isolated using oligo(dT) beads and randomly fragmented by adding fragmentation buffer, followed by cDNA synthesis primed with random hexamers. Next, a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I were added for second-strand synthesis. After end repair, barcode ligation and sequencing adaptor ligation, the double-stranded cDNA library was size selected and PCR amplified. Sequencing was carried out on an Illumina HiSeq platform PE150 with paired-end 150 bp reads.
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| Library strategy |
ssRNA-seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
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| Description |
strand-specific RNA-seq
S48h_CKI3
Sample 42(Batch2)
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| Data processing |
FastQC (v0.11.4, Babraham Bioinformatics) was used to check the quality of raw reads before proceeding with downstream analysis. Trim_galore (v0.3.7, Babraham Bioinformatics) with the parameters: --stringency 5 --paired --fastqc_args was used to trim adaptors and low-quality sequences.
STAR (v2.5.3a) was then applied to align the trimmed reads to the reference genome (hg19, UCSC) with the parameters: --outSAMstrandField intronMotif --outSAMattributes All --outFilterMismatchNmax 10 --seedSearchStartLmax 30 --outSAMtype BAM SortedByCoordinate
Subread (v1.5.2) was used to generate read counts data with the following parameters featureCounts -p -t exon -g gene_id
Differentially expressed genes between treatments were analysed and selected using edgeR (v3.22.3) with false discovery rate (FDR) < 0.05
Removal of unwanted variance (RUVs) package in R was applied to two different batches of transcriptome datasets to eliminate batch variance
Genome_build: hg19, UCSC
Supplementary_files_format_and_content: Tab delimited matrix showing raw counts of refGenes from two batches of raw sequencing samples. Comma separated values containing the combined processed data file generated using RUVs package in R.
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| Submission date |
Jan 28, 2019 |
| Last update date |
May 29, 2019 |
| Contact name |
Thazin Nwe Aung |
| E-mail(s) |
[email protected]
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| Organization name |
University of Adelaide
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| Lab |
Adelson
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| Street address |
Frome
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| City |
Adelaide |
| State/province |
South Australia |
| ZIP/Postal code |
5005 |
| Country |
Australia |
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| Platform ID |
GPL20795 |
| Series (1) |
| GSE125743 |
Fractional Deletion of Compound Kushen Injection Indicates Cytokine Signaling Pathways are Critical for its Perturbation of the Cell Cycle |
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| Relations |
| BioSample |
SAMN10830586 |
| SRA |
SRX5299765 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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