Nextflex small rna library prep kit v3

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.

Small RNA-seq libraries were prepared by using 50 ng of total RNA, isolated from parental cell lines, as input and the NEXTflex Small RNA Library Prep Kit v3 (Bio Scientific) or by Novogene (Hong Kong). Sequencing was performed on an Illumina HighSeq platform at the Deep Sequencing Group (TU Dresden) or Novogene. For mRNA-seq libraries, polyA-RNA was enriched using oligo(dT) beads. Generation of libraries and sequencing were performed by Novogene on an Illumina HiSeq platform. RNA-seq and miRNA-seq data were analyzed as described previously (22 (link),23 (link)). RNA-seq data of the TCGA cohorts were obtained from the GDC portal (https://portal.gdc.cancer.gov).