Pluto Bioinformatics

GSE86337: Transcriptome profiling of 5 human adenocarcinoma cell lines

Bulk RNA sequencing

Purpose: The aim of this study is to compare the transcriptome profiles of a limited number of lung cancer cell lines with the intention of selecting the two most similar cell lines for a mixture experiment (GSE64098).; Methods - Cell Culture: Five lung adenocarcinoma cell lines (H2228, NCI-H1975, HCC827, H838 and A549) from a range of passages (2-4) were grown on 2 separate occasions in RPMI media (Gibco) supplemented with Glutamax and 10\% fetal calf serum to a 70\% confluence. To replicate common experimental conditions cell lines were treated with 0.01\% Dimethyl sulfoxide (Sigma), which is commonly used as a vehicle in drug treatment experiments. After 6 hours of treatment, cells were collected, snap-frozen on dry ice and stored at -80 degree C until required.; Methods - RNA preparation: Total RNA was extracted from between half a million and million cells using Total RNA Purification Kit (Norgen Biotek) according to the kit instructions. RNA quality and concentration were assessed using Nanodrop and Tapestation RNA ScreenTape (Agilent) respectively.; Methods - RNA-seq: 1 ug of total RNA from each sample were used for RNA-seq library preparation using TruSeq Total Stranded RNA with Ribozero (Illumina) according to manufacturer's instructions. Completed libraries were sequenced on HiSeq 2000 with TruSeq SBS Kit v3- HS reagents (Illumina) as 100 bp single end reads at the Australian Genome Research Facility (AGRF), Melbourne. We obtained on average 28 million for each sample (range from 25 to 29 million). Reads were aligned to the human reference genome hg19 using the Rsubread package (version 1.16.1) (Liao et al. 2013). Single reads were then summarized into gene-level counts using FeatureCounts in the reverse-stranded mode (Liao et al. 2014). SOURCE: Matthew Ritchie (mritchie@wehi.edu.au) - The Walter and Eliza Hall Institute of Medical Research