Osmotic stress interferes with DNA damage response and H2AX phosphorylation in human keratinocytes
For total RNA sequencing RNA integrity was estimated with the RNA 6000 Nano Chip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Germany). From total RNA, mRNA was extracted using the NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs, Germany) and RNA-Seq libraries were generated using the NEXTFLEX Rapid Directional qRNA-Seq Kit (Bioo Scientific, Austin) as per the manufacturers recommendations. Concentrations of all samples were measured with a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Germany) and fragment lengths distribution of the final libraries was analyzed with the DNA High Sensitivity Chip on an Agilent 2100 Bioanalyzer (Agilent Technologies). All samples were normalized to 2 nM and pooled equimolar. The library pool was sequenced on the NextSeq500 (Illumina, Germany) with 1x75bp, with 21 to 24 mio reads per sample.