RNA-seq of HPV49
Total RNA was extracted from HPV49 E8- positive cell lines with the RNAeasy kit from Qiagen, including an on-column DNAse digestion step (RNAse free DNAse, Qiagen). The RNA-concentration was measured with the Qubit Fluorometric Quantitation and the RNA Broad-Range Assay (Thermo Fisher Scientific). The RNA-integrity number (RIN) was analysed with the Fragment Analyser 5300 and the fragment analyser RNA-kit (Agilent Technologies) and was sufficient (RIN>8). The mRNA was enriched from 200 ng total RNA with the polyA-purification kit (NEBNext Poly(A) mRNA Magnetic Isolation Module, NEB). The library was prepared using the NEBNext Ultra II Directional RNA-Library-Prep Kit for Illumina (NEB) according to the manufacturers instructions. To determine the molarity, the size of the library (about 400 ng) was analysed with the Fragment Analyzer 5300 and the Fragment Analyzer DNA HS NGS Fragment Kit (Agilent Technologies). The concentration was determined (>5 ng/l) with the Qubit Fluorometric Quantitation and the dsDNA High sensitivity assay (Thermo Fisher Scientific). The library was denatured according to the manufacturers instructions, diluted to 270 pM and sequenced pairwise as paired-end 100 bp reads. The sequencing-platform was an Illumina NovaSeq 6000 with a sequencing-depth of >25 million per sample. The read quality in the fastq-files was assessed with ngs-bits (v.2021_03-101).