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Sample GSM4996452 Query DataSets for GSM4996452
Status Public on Jun 05, 2021
Title CAF3-OLA2
Sample type SRA
 
Source name primary cancer-associated fibroblasts
Organism Homo sapiens
Characteristics disease state: ovarian cancer
cell type: primary cancer-associated fibroblasts (CAFs)
treatment: olaparib 30um for 72h
Treatment protocol When the confluence reach 60-70%, the medium were replaced with new complete medium containing 20uM niraparib or 30uM olaparib and incubated for 72h.
Growth protocol Primary cancer-associated fibroblasts were isolated from tumor specimens and cultured in DMEM/F-12 1:1 medium with 10% FBS and 1×PS in a humidified atmosphere incubator with 5% CO2 at 37 °C.
Extracted molecule total RNA
Extraction protocol Cells in a T75 culture flask were digested and washed with PBS, then dissoved in 1ml trizol and then stored in -80℃ for futher RNA isolation.
A total amount of 2 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using VAHTS mRNA-seq v2 Library Prep Kit for Illumina following manufacturer’ s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using fragmentation buffer. First strand cDNA was synthesized and second strand cDNA synthesis was subsequently performed. Remaining overhangs were converted into blunt ends. After adenylation of 3’ ends of DNA fragments, adaptor with hairpin loop structure were ligated. Then the PCR was performed. At last, Qubit HS quantification, Agilent 2100 Bioanalyzer/Fragment Analyzer 5300 quality control, the final library size of about 350bp.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Data processing The libraries were sequenced on an Illumina NovaSeq platform to generate 150 bp paired-end reads, according to the manufacturer’ s instructions.
Raw data (raw reads) of fastq format were firstly processed through primary quality control. In this step, clean data (clean reads) were obtained by removing read pairs that contain N more than 3 or the proportion of base with quality value below 5 is more than 20%, in any end,or adapter sequence was founded. All the downstream analyses were based on the clean data with high quality.
Paired-end clean reads were aligned to the reference genome using TopHat. We selected TopHat as the mapping tool for that TopHat can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
HTSeq was used to count the reads numbers mapped to each gene.
Differential expression analysis between two conditions was performed using the DEGSeq R package (1.20.0). Differentially expressed genes were defined as those for which the adjusted P-value below 0.05 and the log2(Fold change) more than 1.
Supplementary_files_format_and_content: fpkms
 
Submission date Dec 31, 2020
Last update date Jun 05, 2021
Contact name Tian Fang
E-mail(s) fangtian@hust.edu.cn
Organization name Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology
Lab Cancer Biology Research Center, Key Laboratory of the Ministry of Education
Street address Jiefang Avenue
City Wuhan
State/province Hubei
ZIP/Postal code 430030
Country China
 
Platform ID GPL24676
Series (1)
GSE164088 Gene expression profile of primary CAFs exposed to PARPi or not derived from ovarian cancer patients
Relations
BioSample SAMN17190572
SRA SRX9763467

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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