Nebnext ultra 2 directional rna library prep kit for with purification beads

PolyA NGS library preparation was performed using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina with Purification Beads (NEB #E7760S/L) strictly following the manufacturer's instructions. Sequencing was run on Illumina NovaSeq platform (NovaSeq 6000 SP 300 cycles [2 × 150 bp]) with data output 100 M PE reads/sample. Fastq file processing was done using R package. For bioinformatic analysis, the Bioconductor package edgeR was applied for investigating differential expression. False discovery rate (FDR) was used for multiple testing correction. Unsupervised cluster analysis was done using Genesis 1.8.1 (https://genome.tugraz.at/genesisclient/genesisclient_news.shtml), t-distributed stochastic neighbor embedding (t-SNE) analysis was performed using iDEP.91 (http://bioinformatics.sdstate.edu/idep/). For pathway analysis, gene set enrichment analysis was performed using Rno Reactome pathway gene sets. Gene ontology analysis was performed using Generic GO Term Finder. For network construction, the String database (https://string-db.org/) was applied. For analyzing transcription factor regulatory relationships, TRRUST was used (https://www.grnpedia.org/trrust/). Raw data was uploaded to NCBI GEO database (GSE202934).