Detection of SINEUP RNA modification by Oxford Nanopore direct-RNA sequencing

To detect the modifed bases in SINEUP RNA, we compared chemically modified in vitro transcribed (IVT) SINEUP-GFP RNA and in-cell transcribed (ICT) SINEUP RNA from SINEUP-GFP and sense EGFP co-transfected HEK293T/17 cells. Comparative study of Nanopore direct RNA sequencing data from non-modified and modified IVT samples against the data from ICT SINEUP RNA sample revealed modified k-mers positions in SINEUP RNA in the cell. Overall design: We used in vitro transcribed (IVT) SINEUP-GFP RNA with 0 % (no modification) and 20% ? modification conditions. In parallel, we captured and enriched in-cell transcribed (ICT) SINEUP RNA from SINEUP-GFP and sense EGFP co-transfected HEK293T/17 cells by SINEUP-specific biotinylated oligos. Both in vitro and in-cell transcribed samples were sequenced by Nanopore direct RNA sequencing.