HD-MB03 cells were treated with DMSO or 1 nM dinaciclib for 24 h. All treatment conditions were submitted and processed in triplicates at the Sequencing facility of the Cancer Research, UK. Sequencing libraries were prepared by using the QuantSeq kit (Lexogen) and run on an Illumina NextSeq sequencer.
A sequential alignment procedure was performed to map raw reads against the GRCh38 Human Genome reference (release 83). First, all reads were aligned by ‘STAR’54 (link); then, using ‘Bowtie2’55 (link), we locally mapped those reads discarded by STAR. Gene expression quantification was computed by ‘featureCounts’56 (link). The ‘DaMiRseq’ Bioconductor/R package was used to filter out genes (less than 10 counts in more than 50% of samples), perform the normalization (variance stabilizing transformation), and search for confounding factors57 (link). Differential analysis was performed by the ‘limma’ R/Bioconductor package58 (link). A gene was deemed significant whether the P value was < 0.05 and logFC threshold of 0.5. A Benjamini–Hochberg procedure was performed to control the false discovery rate (FDR).
A sequential alignment procedure was performed to map raw reads against the GRCh38 Human Genome reference (release 83). First, all reads were aligned by ‘STAR’54 (link); then, using ‘Bowtie2’55 (link), we locally mapped those reads discarded by STAR. Gene expression quantification was computed by ‘featureCounts’56 (link). The ‘DaMiRseq’ Bioconductor/R package was used to filter out genes (less than 10 counts in more than 50% of samples), perform the normalization (variance stabilizing transformation), and search for confounding factors57 (link). Differential analysis was performed by the ‘limma’ R/Bioconductor package58 (link). A gene was deemed significant whether the P value was < 0.05 and logFC threshold of 0.5. A Benjamini–Hochberg procedure was performed to control the false discovery rate (FDR).
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