title: TERA-Seq of HeLa: Cap & 5P-Poly(A)
design: 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. The quality of RNA was assessed on an Agilent Bioanalyzer. Depending on downstream application, RNA was subjected to either oligo-dT selection or to ribosomal RNAs and abundant small, non-coding RNAs depletion from total RNA using custom biotinylated DNA oligos. RNA was further subjected to various treatments and its ends were marked by adapter ligation to the 5' (5TERA), 3' (TERA3) or both ends (5TERA3). For 5TERA; to select 5' monophosphate (5P)-containing RNAs, poly(A) RNA was ligated to a 5' biotinylated long adapter (5' adapter) or to a 5' short adapter to generate 5P-Poly(A) and 5P short-Poly(A) libraries respectively. To select for both 5P and 5' hydroxyl RNAs, poly(A) RNA was treated with T4 PNK before ligation to the 5' short adapter (5P short-PNK-Poly(A) library). For the selection of only capped RNAs, poly(A) 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) library). To capture both capped and 5P-containing RNAs, poly(A) RNA was only treated with RppH followed by 5' adapter ligation (5P & Cap-Poly(A) library). For TERA3; 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 of ligated RNA by 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 by Streptavidin beads. All samples (12) were subsequently subjected to direct RNA nanopore sequencing library preparation (SQK-RNA001 and SQK-RNA002, ONT). Sequencing was performed on MinION device using R9.4.1 flow cell (FLO-MIN106) for 48 h or 72 h run time.