TERA-Seq of HeLa: CTRL-poly(A)
TERA-Seq library preparation for direct RNA sequencing using Oxford Nanopore Technologies (ONT) library kits. Total RNA was extracted from HeLa cells after immediate cell lysis with Trizol, and its integrity was assessed on a Bioanalyzer prior to each library preparation. Libraries were prepared using nanopore direct RNA sequencing kits (SQK-RNA001 and SQK-RNA002) following manufacturers instructions for the CTRL-Poly(A) library or with modifications as follows. Total RNA was subjected to either oligo-dT selection or to ribosomal RNAs and abundant small, non-coding RNAs depletion using custom biotinylated DNA oligos. RNA was further subjected to various enzymatic treatments and its ends were marked by adapter ligation to the 5' end (5TERA), 3' end (TERA3) or both ends (5TERA3). For 5TERA library generation; to identify 5' monophosphate (5P)-containing RNAs, poly(A)-enriched RNA was ligated to a 5' biotinylated long adapter (5' adapter; 5P-Poly(A)) or to a 5' short adapter (5P short-Poly(A)). To identify both 5P- and 5' hydroxyl (5OH)-containing RNAs, poly(A)-enriched RNA was treated with T4 PNK before ligation to the 5' short adapter (5P short-PNK-Poly(A)). To identify 5OH-containing RNAs, poly(A)-enriched RNA was ligated to the 5' adapter using RtcB ligase. To identify only capped RNAs, poly(A)-enriched RNA was dephosphorylated using Quick Calf Intestinal Phosphatase. The 5' cap of RNA was then removed using RNA 5' Pyrophosphohydrolase (RppH) followed by ligation to the 5' adapter (Cap-Poly(A)). To identify both capped and 5P-containing RNAs, poly(A)-enriched RNA was only treated with RppH followed by ligation to the 5' adapter (5P & Cap-Poly(A)). For TERA3 library generation; 3 biological replicates were prepared using depleted total RNA that was ligated to a 3' adapter. To simultaneously capture the 5' and 3' ends of RNAs (5TERA3); 3 biological replicates were prepared using depleted total RNA that was first subjected to decapping using RppH, ligation to the 5' adapter, enrichment on streptavidin beads, and ligation to the 3' adapter.  To prepare a library from synthetic SIRV E2 transcripts, RNA was first selected on oligo-dT beads and was treated with RppH. The RNA was ligated to the 5' adapter followed by enrichment of ligated RNA on streptavidin beads. Sequencing was performed on MinION device using R9.4.1 flow cell (FLO-MIN106) for 48 h or 72 h.