Nanopore direct RNA sequencing and data analysis

8x10⁷ cells from undifferentiated or methylcellulose differentiated keratinocytes containing HPV18 (WT or ΔCTCF) samples for RNA extraction using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions and DNaseI treated (Promega). 500 ng of polyA+ RNA was used in conjunction with the direct RNA sequencing kit (Oxford Nanopore technologies, Oxford, UK [SQK-RNA002]). All protocol steps are as described in [37]. The reads were aligned to the human (GRCh37) and HPV18 ({"type":"entrez-nucleotide","attrs":{"text":"AY262282.1","term_id":"30172004","term_text":"AY262282.1"}}AY262282.1) genomes using minimap2 [38] with options “-ax splice -uf -k14” for nanopore direct RNA mapping. The splicing coordinates were extracted from the bam files using custom scripts. HPV18 transcripts were included in the dataset when a minimum threshold of three reads per million in at least two samples was achieved to ensure that each transcript was identified at least four times in multiple samples. Illumina and Nanopore data sets used in this study are available at the European Nucleotide Archive (http://www.ebi.ac.uk/ena/data/view/PRJEB47821).