title: MinION sequencing
library_construction_protocol: RNA was transcribed from PCR templates LacZ with T7 RNAP, cleaned on KAPA Pure Beads (Roche; cat. no: KK8001) and examined on denaturing gel. 500 ng of RNA was polyadenylated in presence rNTPs in ratio CTP:GTP:UTP:ATP 1:3.3:2.7:23 (Traut 1994) by Escherichia coli PAP polymerase (New England Biolabs; cat. no: M0276) in 20 µl reaction for 30 min at 37°C, according to manufacturer's protocol. The reaction was stopped with EDTA pH 8.0 (final 10 mM), purified RNA was measured with Qubit RNA high sensitivity (ThermoFischer Scientific; cat. no: Q32852) protocol, visualized by denaturing electrophoresis, and used to prepare libraries with a Direct RNA Sequencing Kit (SQK-RNA002) from Oxford Nanopore Technologies (ONT), following the manufacturer's instructions. Sequencing was performed using R9.4.1 RevD flow cells on a MinION device, controlled by MinKNOW software (ONT) as described by Bilska et al. (2020).