GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5687759

Query DataSets for GSM5687759

Status

Public on Aug 05, 2023

Title

DU145_RCAN3_NC

Sample type

SRA

Source name

prostate cancer cells

Organism

Homo sapiens

Characteristics

cell line: DU145 prostate cancer cell
treatment: transfected with negative control plasmid

Extracted molecule

total RNA

Extraction protocol

Total RNAs were extracted from samples using TRIzol reagent(Invitrogen, cat. NO 15596026) following the methods by Chomczynski et al (DOI:10.1006/abio.1987.9999). DNA digestion was carried out after RNA extraction by DNaseI. RNA quality was determined by examining A260/A280 with NanodropTM OneCspectrophotometer (Thermo Fisher Scientific Inc). RNA Integrity was confirmed by 1.5% agarose gel electrophoresis. Qualified RNAs were finally quantified by Qubit3.0 with QubitTM RNA Broad Range Assay kit (Life Technologies，Q10210).
2 μg total RNAs were used for stranded RNA sequencing library preparation using Ribo-off rRNA Depletion Kit (Human/Mouse/Rat) (Catalog NO. MRZG12324, Illumina) and KC-DigitalTM Stranded mRNA Library Prep Kit for Illumina® (Catalog NO. DR08502, Wuhan Seqhealth Co., Ltd. China) following the manufacturer’s instruction. The kit eliminates duplication bias in PCR and sequencing steps, by using unique molecular identifier (UMI) of 8 random bases to label the pre-amplified cDNA molecules. The library products corresponding to 200-500 bps were enriched, quantified and finally sequenced on NovaSeq 6000 sequencer (Illumina) with PE150 model.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

SKT3_All_samples_rpkm.xls

Data processing

Raw sequencing data was first filtered by Trimmomatic (version 0.36), low-quality reads were discarded and the reads contaminated with adaptor sequences were trimmed. Clean Reads were further treated with in-house scripts to eliminate duplication bias introduced in library preparation and sequencing. In brief, clean reads were first clustered according to the UMI sequences, in which reads with the same UMI sequence were grouped into the same cluster. Reads in the same cluster were compared to each other by pairwise alignment, and then reads with sequence identity over 95% were extracted to a new sub-cluster. After all sub-clusters were generated, multiple sequence alignment was performed to get one consensus sequence for each sub-clusters. After these steps, any errors and biases introduced by PCR amplification or sequencing were eliminated.
Deduplicated Reads were mapped to the reference genome of human using STRA software (version 2.5.3a) with default parameters.
Reads mapped to the exon regions of each gene were counted by featureCounts (Subread-1.5.1; Bioconductor) and then RPKM was calculated.
Genes differentially expressed between groups were identified using the edgeR package (version 3.12.1). p-value cutoff of 0.05 and Fold-change cutoff of 2 were used to judge the statistical significance of gene expression differences.
Gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis for differentially expressed genes were both implemented by KOBAS software (version: 2.1.1) with a P-value cutoff of 0.05 to judge statistically significant enrichment. Alternative splicing events were detected by using rMATS (version 3.2.5) with an FDR value cutoff of 0.05 and an absolute value of Δψ of 0.05.
Genome_build: hg38
Supplementary_files_format_and_content: reads count
Supplementary_files_format_and_content: rpkm value

Submission date

Nov 12, 2021

Last update date

Aug 05, 2023

Contact name

Gang Luo

Organization name

Huazhong University of Science and Technology

Street address

26 Shengli Street

City

Wuhan