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Sample GSM4962598

Query DataSets for GSM4962598

Status

Public on Jan 12, 2021

Title

LNCaP cell line - 24h PDA-66 rep1

Sample type

SRA

Source name

Prostate carcinoma cells

Organism

Homo sapiens

Characteristics

cell line: LNCaP
cell type: prostate adenocarcinoma cell line derived from lymph node metastasis
treatment: treated with 15 µM PDA-66 for 24 h

Treatment protocol

Cells were treated with 15 µM PDA-66 for 12 h or 24 h with 3 replicates per cell line and incubation period. Control cells were treated with 0.15% (v/v) DMSO as PDA-66 is dissolved in DMSO.

Growth protocol

PC-3 cells were cultivated in DMEM/Ham´s F-12 medium (Biochrom GmbH, Berlin, Germany). LNCaP cells were grown in RPMI 1640 medium (Biochrom GmbH). CT1258 cells were cultivated in medium 199 (Live Technologies GmbH, Darmstadt, Germany). All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS Superior, Biochrom GmbH) and 2% penicillin/streptomycin (Biochrom GmbH). All cells were cultivated at 37 °C in a humidified atmosphere of 5% CO2.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using the AllPrep DNA/RNA Mini Kit (Qiagen GmbH, Hilden, Germany) in accordance with the manufacturer’s protocol. RNA integrity numbers (RIN) were determined using a Bioanalyzer 2100 (Agilent Technologies Inc., Santa Clara, CA, USA).
Sequencing libraries were prepared using 1 μg total RNA with RIN > 7. PolyA RNA was enriched and ligated to sequencing adapters using the NEBNext Ultra RNA preparation kit (New England Biolabs Inc., Ipswich, MA, USA) in accordance with the manufacturer’s protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

L24-1

Data processing

For the human PC-3 and LNCaP cells, mapping and read counting were conducted using the RNA Express (version: 1.0.0) workflow within the Illumina basespace environment. In brief, after mapping to the human HG19 reference genome using the STAR (2.3.1s) aligner read counts for 23710 annotated RefSeq genes were generated. For CT1258, sequences were aligned to the canine genome (Broad CanFam3.1/canFam3, Sep. 2011) using the Burrows-Wheeler Aligner (BWA). For each of the 24,581 annotated protein-coding canine genes (EMBL gene ID nomenclature), the aligned reads were counted using the R package GAGE.
Differential gene expression analyses for canine and human samples were conducted using the BioconductorR package edgeR 3.14.0. The average total read counts mapped within annotated genes were 12.2M (STD: 0.7M), 15.8M (STD: 1.1M), 13.5M (STD: 0.9M) for CT1258, PC-3 and LNCaP samples, respectively. Each PDA-66 treatment group (12 h or 24 h) was compared to the respective control cells treated with DMSO.
After multidimensional scaling and plotting of the data, one PC-3 DMSO treated control sample (PD1) was identified as outlier and was excluded from further analyses so that the PC-3-24 h-DMSO control group consisted of only two independent samples.
Genes with a false discovery rate adjusted p-value (FDR) of less than 0.001 were considered to have significantly different expressions compared to the control samples. These genes were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for functional enrichment analyses using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Functional Annotation Tool.
Genome_build: HG19 and CanFam3.1
Supplementary_files_format_and_content: edgeR gene counts for the three cell lines
Gene tables with fold changes and FDRs for every treatment vs control

Submission date

Dec 08, 2020

Last update date

Jan 12, 2021

Contact name

Jan Torben Schille

Organization name

University of Veterinary Medicine Hannover

Department

Small Animal Clinic

Street address

Buenteweg 9