RNA-Seq

Three tumors were pooled per condition. Tumors were grinded into a fine powder in liquid nitrogen using a mortar and pestle. RNA was extracted using the RNeasy Mini Plus kit (Qiagen) following the manufacturer’s instructions with the optional on-column DNase digestion. Quality of the extracted RNA samples was assessed using the Agilent Bioanalyzer (Agilent, Santa Clara, CA, USA). All samples had a RNA Integrity Number (RIN) > 8.7. Library preparation and sequencing were performed at the McGill University and Génome Québec Innovation Centre using the NEB/KAPA stranded mRNA lib prep kit and sequenced on an Illumina HiSeq (Illumina, San Diego, CA, USA). Fastq files were verified for quality using FastQC (v0.11.4) and trimmed using Trimmomatic (v0.32) (with TRAILING:30) to remove adaptors and portions of low-quality reads. Read pairs were then aligned to the human genome build hg38 using an annotation file obtained from Ensembl (v 87) with the splice-aware RNA-seq aligner STAR (v2.5.1)⁷⁶ using default parameters. Reads were also aligned separately on the Gallus gallus-5.0 genome assembly with the annotation file obtained from Ensembl (v 87) to verify the degree of contamination of the samples with egg tissue. Aligned reads were then assigned to genomic features using featureCounts from Subread (v1.5.2)⁷⁷. FeatureCounts parameters were set to (-C,–largestOverlap, -p, -B, -s1,–minOverlap 10, -M). Count files produced by featureCounts were then compared between controls and knockdowns for SW480 and HCT116 cells separately for differential expression analysis using DESeq. 2 (v1.10.1)⁷⁸. GSEA analyses were performed using the open source software (GSEA, v2.0, Broad Institute, MA, USA)⁷⁹.