Gene expression data related to Broche et al.: "Genome-wide deposition of 6-methyladenine in human DNA reduces the viability of HEK293 cells and directly influences gene expression"

Total RNA was isolated from ~10^6 cells using the RNeasy® Mini Kit (Qiagen), and quantified via NanoDrop1000. For each library, 1 µg RNA was taken as input material. Since total RNA is mostly constituted of rRNA (≥ 80%), poly-adenylated mRNA was first enriched using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB).

Afterwards, RNA-seq libraries were generated using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB), in combination with NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1) (NEB), thus resulting in strand-specific RNA libraries (opposite sense strand). For quality assessment of the RNA-seq libraries, fragment size distributions and concentrations were determined by Labchip® GX Touch (Perkin Elmer).

Finally, 300 ng of each RNA library was submitted to Novogene Corporation Inc. U.K. for Next-Generation-Sequencing (Illumina Novaseq 6000 system, paired-end, 2 x 150bp-mode). Sequencing data were received in FASTQ format (~2 x 20 million reads/sample), and all subsequent data processing steps were performed on the European Galaxy Platform (Afgan et al., 2016).