title: IVT RNA betagal construct with templated poly(A) tails, produced by wild-type T7 RNAP, direct RNA sequencing
library_construction_protocol: RNA was transcribed from PCR templates LacZ with wild-type T7 RNAP. RNA was prepared at 37°C for 1.5 h in 50 µl reaction mix containing: 600 pmols template, 1× buffer (200 mM Tris-HCl, 30 mM MgCl2, 10 mM spermidine, 50 mM NaCl), 5 µl of rNTPs mix (20 mM each), 5 µl of 100 mM DTT, 0.5 µl of 1% Triton X-100, 80U ribonuclease inhibitor and homemade T7 RNAP. RNA was cleaned on KAPA Pure Beads (Roche; cat. no: KK8001), concentration was assessed with Qubit RNA high sensitivity (ThermoFischer Scientific; cat. no: Q32852) protocol. 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).