Total RNA was extracted from each cell using RNeasy Plus (QIAGEN) after 168 h siRNA transfection, and the quantity and quality was determined using Bioanalyzer (Agilent Technologies). Single read sequencing was performed with NextSeq 500 (Illumina) using 75 cycles High Output kit v2. Sequences were trimmed for sequencing adapters and low quality 3' bases using Trimmomatic version 0.35 [3 (link)] and aligned to the reference Chlorocebus sabaeus genome version ChlSab1.1 (gene annotation from Ensembl 104) or Homo sapiens genome version GRCh38 (gene annotation from Gencode version 37) using STAR version 2.7.1a [7 (link)].
Raw gene expression counts were obtained directly from STAR, and normalization and differential expression analysis were performed using DESeq2 version 1.30.1 [24 (link)]. Metascape software was used for gene ontology analysis [56 (link)]. We used unadjusted p-values for U2OS analysis as false discovery rate calculation has low power in datasets with small effect sizes (i.e. low fraction of changed genes in RNAseq) [22 (link)], and in our dataset Benjamini–Hochberg procedure resulted in over-represented p.adjusted values (Additional file2 : Table S1). For comparison analyses between COS-7 and U2OS, gene identifiers were converted from C. sabaeus to H. sapiens using the R/Bioconductor package biomaRt (version 2.48.3).
Raw gene expression counts were obtained directly from STAR, and normalization and differential expression analysis were performed using DESeq2 version 1.30.1 [24 (link)]. Metascape software was used for gene ontology analysis [56 (link)]. We used unadjusted p-values for U2OS analysis as false discovery rate calculation has low power in datasets with small effect sizes (i.e. low fraction of changed genes in RNAseq) [22 (link)], and in our dataset Benjamini–Hochberg procedure resulted in over-represented p.adjusted values (Additional file
Free full text: Click here
