RNA-seq analysis with rRNA depletion

RNA extraction was performed as previously described^{4 (link)}. Following extraction, ribosomal RNA (rRNA) was depleted from the total RNA samples using the RiboErase module of the KAPA RNA Hyper+RiboErase HMR Kit (Roche Cat. No. 08098140702). The rRNA-depleted RNA was then applied for library preparation. The procedure was conducted following the protocol provided with the KAPA RNA Hyper+RiboErase HMR Kit. The prepared libraries were validated for quality and quantification using the Agilent 2100 Bioanalyzer. The libraries were then sequenced on an Illumina NovaSeq 6000, utilizing a paired-end sequencing strategy (2 × 150 nucleotide read length with sequence depth of 15–20M paired reads).
RNA-Seq data was handled via Kallisto (version 0.46.1)^{62 (link)}. R package EdgeR (edgeR_3.39.6) was used to generate normalized and filtered read counts (counts >10)^{63 (link)}. Differential expression was performed using Limma-Voom (limma_3.53.10)^{64 (link)}. R package fgsea (fgsea_1.24.0) was used for Gene Set Enrichment Analyis (GSEA). Further R packages tidyverse, gtable, gplots, ggplot2 and EnhancedVolcano (EnhancedVolcano_1.15.0) were also used for generating figures.