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Sample GSM5508599 Query DataSets for GSM5508599
Status Public on Aug 08, 2021
Title SK-EPI-siBCRP-2
Sample type SRA
Source name SK-BR-3 cell line
Organism Homo sapiens
Characteristics cell type: breast cancer
cell line: SK-BR-3
tissue: breast
knock down: siBCRP
Treatment protocol For knockdown of BCRP, cells were treated with siNC or siBCRP siRNAs using Lipofectamine RNAiMax for 48 h, then the RNA was isolated by Trozol reagent.
Growth protocol Human breast cancer cell lines SK-BR-3 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The epirubicin-resistant cell lines (SK/EPI) were established based SK-BR-3 cells by long-time multistage induction using an increasing concentration of epirubicin.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using Trizol reagent following the manufacturer’s instructions.
A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) 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 divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H - ). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 m in followed by 5 min at 95 °Cbefore PCR. Then PCR was performed with Phusion High -Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Description SK-EPI-sINC_SK-EPI-siBCRP.txt
Data processing Reads mapping to the reference genome: Hisat2 v2.0.5
Quantification of gene expression level: featureCounts v1.5.0-p3
Differential expression analysis: DESeq2 R package (1.16.1)
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text file includes FPKM value for each sample
Submission date Aug 07, 2021
Last update date Aug 09, 2021
Contact name He Zhang
Phone +861360204722
Organization name Tianjin Medical University Cancer Institute and Hospital
Department Public Laboratory
Street address Tianjin 300060
City Tianjin
State/province Tianjin
ZIP/Postal code 300060
Country China
Platform ID GPL24676
Series (1)
GSE181632 Mitochondrial BCRP sustains the proliferation and survival of drug-resistant breast-cancer cells by regulating intracellular ROS
BioSample SAMN20606419
SRA SRX11668309

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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