title: IVT RNA Renilla luciferase constructs with various poly(A) tail compositions, input for neural network training, direct RNA sequencing
library_construction_protocol: Set of transcripts with well-defined poly(A) tails were prepared in vitro by T7 RNAP. Constructs contain transcript body comprising a fragment of Renilla luciferase and a variant of poly(A) tail introduced on specific PCR primers (either blank or with singular C/G/U inclusion in the middle of the 60nt poly(A) tail). 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).