MinION sequencing; Nanopore direct RNA sequencing of poly(A) spike-in with 90 adenosines (Rluc template)
Templates for spike-ins were prepared in two consecutive PCR reactions. First, 827-bp fragment of Renilla luciferase from pRL5Box plasmid was amplified using RLucF1 and RLucR1 primers. Purified amplicon was used as a template in the second PCR reaction with RLuc_T7_F2 primer containing the T7 promoter sequence, and backward primer RLuc_Ax_R2 introducing poly(A) tail of a defined length (from 10 to 90 As). The resulting PCR product was analyzed and purified by gel electrophoresis. In vitro transcription reaction was performed at 37°C for 1.5h in a 50µl reaction volume containing: 600 pmols T7 template, 10µl of 5x transcription buffer (200mM Tris-HCl, 30mM MgCl2, 10mM spermidine, 50mM NaCl), 5µl of rNTPs mix (20mM each), 5µl of 100mM DTT, 0.5µl of 1%Triton X-100, 80U Ribonuclease Inhibitor, 100U T7 RNA polymerase. Then, DNA template was digested by adding 4U of TURBO DNase (Ambion) for the next 15min. The reaction was stopped with 2.5ul of 0.5M EDTA pH8.0 and following phenol/chlorophorm extraction and ethanol precipitation. The quality of spike-ins RNA was visually assessed by denaturing electrophoresis. Spike-ins were purified on RNA purification beads (Kapa Pure beads) and subjected to nanopore sequencing. Nanopore direct-RNA libraries were prepared with Direct RNA Sequencing Kit (Oxford Nanopore Technologies, SQK-RNA002) using 150 ng of Saccharomyces cerevisiae oligo(dT)-enriched mRNA, mixed with high-quality RNA from other species (mouse) and 50ng of spike-in RNA. Sequencing was performed with MinION device (Oxford Nanopore Technologies, Flow cell type FLO-MIN106; RevD) and basecalled with Guppy (Oxford Nanopore Technologies)