Libraries for RNA-sequencing (RNA-seq) were essentially prepared according to the manufacturer’s protocols. For total RNA-seq, 1 μg of total RNA served as input for ribosomal RNA depletion using RiboCop v1.2 (Lexogen). The Ultra Directional RNA Library kit (NEB) was used for library generation. Sequencing was performed on an Illumina NextSeq 500 platform. For the generation of small RNA-seq libraries, 50 ng of total RNA served as input using the NEXTflex Small RNA Library Prep Kit v3 (Bio Scientific). Sequencing was performed on the Illumina HighSeq 2000 platform.
For RNA-seq data analyses, low quality read ends as well as remaining parts of sequencing adapters were clipped off using Cutadapt (v 1.14). For total and small RNA-seq analyses, reads were aligned to the human genome (UCSC GRCh38) using HiSat2 (v 2.1.0; (32 (link))) or Bowtie2 (V 2.3.2; (33 (link))), respectively. FeatureCounts (v 1.5.3; (34 (link))) was used for summarizing gene-mapped reads. Ensembl (GRCh38.89; (35 (link))) or miRBase (v 21; (36 (link))) was used for annotations. Differential gene expression (DE) was determined by the R package edgeR (v 3.18.1; (37 (link))) using TMM normalization.
For RNA-seq data analyses, low quality read ends as well as remaining parts of sequencing adapters were clipped off using Cutadapt (v 1.14). For total and small RNA-seq analyses, reads were aligned to the human genome (UCSC GRCh38) using HiSat2 (v 2.1.0; (32 (link))) or Bowtie2 (V 2.3.2; (33 (link))), respectively. FeatureCounts (v 1.5.3; (34 (link))) was used for summarizing gene-mapped reads. Ensembl (GRCh38.89; (35 (link))) or miRBase (v 21; (36 (link))) was used for annotations. Differential gene expression (DE) was determined by the R package edgeR (v 3.18.1; (37 (link))) using TMM normalization.