Total RNA from cultured cells was isolated with the QIAGEN RNeasy Mini Kit with on-column DNase digestion. RNA quality checks were performed with an Agilent 2100 Bioanalyzer (Eukaryotic Total RNA Nano kit). Library preparation (500 ng input RNA) was performed with the NEBNext Poly(A) mRNA Magnetic Isolation Module (#E7490) with SPRIselect Beads (Beckman Coulter), the NEBNext Ultra II Single-End RNA Library Prep kit (#7775S), and the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) according to the manufacturer’s instructions. Library size was confirmed with an Agilent 2100 Bioanalyzer (DNA1000 chip). Pooled libraries were diluted to 1.8 pM (concentrations checked with the Qubit Fluorometer high-sensitivity assay, Thermo Fisher), and sequenced on an Illumina NexSeq 500 instrument with the NexSeq 500 75-cycle high-output kit.
For data analysis, FASTQ files were generated with the bcl2fastq command line program (Illumina). Transcript alignment was performed with Salmon (Patro et al., 2017 (link)). Differential expression analysis (NB4A- vs. DMSO-treated cells) was performed with the DESeq2 R package. DESeq2 “stat” values for each gene were used as inputs to pre-ranked GSEA, where enrichment was tested against the Hallmark gene sets from the Molecular Signatures Database (MSigDB). Access to sequencing data is discussed in the data availability section.
For data analysis, FASTQ files were generated with the bcl2fastq command line program (Illumina). Transcript alignment was performed with Salmon (Patro et al., 2017 (link)). Differential expression analysis (NB4A- vs. DMSO-treated cells) was performed with the DESeq2 R package. DESeq2 “stat” values for each gene were used as inputs to pre-ranked GSEA, where enrichment was tested against the Hallmark gene sets from the Molecular Signatures Database (MSigDB). Access to sequencing data is discussed in the data availability section.