RNA-Seq Analysis of Ascites Cells

RNA was extracted from ascites cells using the Norgen Total RNA extraction kit (cat. # 17200, Norgen, Thorold, ON, Canada). Input was normalized based on cell count. RNA integrity numbers were measured using BioAnalyzer (Agilent, Santa Clara, CA, USA) and ranged from 7.1 to 9.9 (with one outlier of 2.9, which was removed from further analysis). RNA was quantified using Qubit (Thermofisher). Then, 800 ng of total RNA was used as input to the NEBNext Ultra RNA Library Prep Kit for Illumina using the polyA selection method (E7530S and E7490S). Libraries were barcoded using NEBNext multiplex oligos for Illumina (E7335S). Five samples per lane were pooled on an Illumina HiSeq 2500 flow cell and sequenced an average of 29M aligned reads per sample and an average quality score of 35, with 93% >Q30. Samples were processed using our published primary analysis tool, aRNApipe.^{16 (link)} Count tables were processed using DESeq2^{17 (link)} for differential expression analysis. No clinical covariates were used in the model since the experiment was all done in vitro. R was used for all additional statistical analysis. Hierarchical clustering was done using the heatmap.2 function in the gplots R package.