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Sample GSM4845008

Query DataSets for GSM4845008

Status

Public on Dec 15, 2020

Title

A2780 cells untreated - rep 2

Sample type

SRA

Source name

ovarian cancer cell line

Organism

Homo sapiens

Characteristics

growth phase: Log phase cells
cell line: ECACC 93112519
treatment: untreated

Treatment protocol

The process of resistance development was commenced from treatment A2780 parental cell line with 0.25 nM PTX for 7 days. After this time cells reached the stable growth in this drug concentration and the first subline was generated - A/0.25PTX-7d. In the next steps PTX dose was dobled until the A/2PTX-31d cell subline was generated, which was the parental cell line for the first NGS- analyzed cell line - A/4PTX-63d. A2780 cells and 6 sublines were cultured in duplicate in 25 cm2 flasks without PTX for 3-4 days before total RNA isolation. Then medium was removed, cells were rinsed two times with phosphate buffered saline (PBS) and the cultures were terminated by adding RLT buffer and collecting cell lysate with a cell scraper. Cell extracts were frozen at -80°C or used immediately.

Growth protocol

Cells were grown in monolayer and conventionally were maintained in RPMI 1640 medium with phenol red , supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Life Technologies – Thermo Fisher Scientific), without antibiotics and antimycotics, at 37ºC and 5% CO2.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from 5x106 cells using RNeasy Mini Kit (Qiagen) according to manufacturer instructions. Method is based on lysing cells in RLT buffer containing guanidine isothiocyanate, purification the product on spin column with silica-gel membrane and treatment by DNaseI to remove genomic DNA contamination. Isolated RNA was stored in -80ºC.
RNA integrity number (RIN) was analyzed using Bioanalyzer 2100 and RNA 6000 Nano Kit (Agilent Technologies), according to manufacturer instructions. RNA concentration was measured using fluorometer Qubit and Qubit RNA BR Assay Kit (Life Technologies), according to manufacturer instructions. Fourteen NGS libraries were prepared following the protocol of the TrueSeq RNA Sample Preparation Kit v2 (Illumina). From 4 μg of total RNA input of each sample the poly-A containing mRNA molecules were purified using oligo-dT attached magnetic beads. Then the mRNA was fragmented into about 150 nucleotide (nt) pieces using divalent cations under 65ºC. The cleaved mRNA fragments were primed with random hexamers and reverse transcribed by SuperScript III reverse transcriptase into first strand cDNA. Subsequent stage involved second strand cDNA synthesis using DNA Polymerase I and removing the RNA template using RNaseH. Double stranded cDNA (ds cDNA) was purified using AMPure XP beads. Then the overhangs resulting from fragmentation were converted into blunt ends, phosphorylated on 5’ ends and a single A nucleotide was added to 3’ ends. Multiple indexing adapters with single T nucleotide 3’ ends were ligated to the ends of the ds cDNA. Finally, the 12 cycle PCR was performed to selectively enrich those DNA fragments that had adapter molecules on both ends and to amplify the amount of DNA to sequence. Libraries were qualified using Bioanalyzer 2100 and DNA 1000 Kit (Agilent), according to manufacturer instructions. In all libraries the final product was an anticipated band at approximately 270 bp. The total concentration of the libraries was measured using fluorometer Qubit and Qubit dsDNA BR Assay Kit (Life Technologies), according to manufacturer instructions. Libraries were additionally quantified with qPCR according to Illumina recommended protocol, using Rotor-Gene Q (Qiagen) and KAPA SYBR FAST qPCR Master Mix Universal (Kapa Biosystems). Validated libraries were diluted to 10 nM concentration, then pooled in batches of two differentially indexed samples in equal amounts, denatured and diluted to 10 pM concentration. Cluster generation was performed in cBot (Illumina), where the libraries were bound with a flow cell (two libraries per lane). Sequencing by synthesis (SBS) was performed with Genome Analyzer IIx (Illumina). Technical replicates of the libraries were clustered and sequenced with the use of different flow cell, but with the same 75-cycle multiplexed single-read sequencing recipe. Finally, we obtained approximately 25 million 75 nt-long reads per library.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Description

A2780b

Data processing

Sequencing by synthesis (SBS) was performed with Genome Analyzer IIx (Illumina). Technical replicates of the libraries were clustered and sequenced with the use of different flow cell, but with the same 75-cycle multiplexed single-read sequencing recipe. Finally, we obtained approximately 25 million 75 nt-long reads per library.
Raw reads were next filtered according to the quality with the use of FASTX-Toolkit. Reads consisted of less than 80% nucleotides with at least 28 quality were discarded. On average 15% reads were filtered out per library.
The remaining reads were mapped to the human reference genome version hg19 with TopHat2. On average, 96% of reads remained after filtering were mapped to the genome.
After aligning reads, the number of reads (counts) mapped to known genes were obtained with usage of featureCounts function implemented in R package “Rsubread”. All the genes with very low expression values across the examined samples were discarded – only genes with more than 5 normalized reads mapped to the gene for at least 2 samples were included in further analysis.
The differential expression analysis was performed using linear models implemented in ‘limma’ package for R. The counts for every gene were normalized with voom function which calculates the log2 value of counts, estimates their mean and variance, and generates a precision weight for each observation. Next, the linear model was fitted to the data set including expression values for each gene (using lmfit function) and empirical Bayes statistics (eBayes function) was used to improve power of a test used for differential expression detection in sets with a high number of genes and a small number of replicates.
Genome_build: human reference genome version hg19
Supplementary_files_format_and_content: Matrix table coma delimited with raw gene counts for every gene and every sample

Submission date

Oct 21, 2020

Last update date

Dec 15, 2020

Contact name

Aleksandra Maria Swiercz

E-mail(s)

aswiercz@cs.put.poznan.pl

Organization name

Poznan University of Technology

Department

Institute of Computing Science