RNA-seq of Escherichia phage T7: T7 genome expressed in purified T7 RNA polymerase
The T7 genome was expressed using purified T7 RNA polymerase (TranscriptAid T7 High Yield Transcription Kit, Thermo Fisher #K0441). After a 2-hour incubation at 37C, the RNA was treated with DNase I (2 L) for 15 minutes at 37C, followed by 2 L 0.5 M EDTA (pH 8.0) and incubation at 65C for 10 minutes to stop the reaction. RNA was then purified using the Monarch RNA Cleanup Kit (NEB T2050). Polyadenylation was performed according to the ONT protocol (v4, March 2023) with E. coli Poly(A) Polymerase (NEB M0276L). RNA libraries were prepared using the Direct RNA Sequencing Kit (SQK-RNA004, ONT) without amplification or selection. Sequencing was conducted on a MinION Mk1B (ONT) with a Flow Cell RNA (FLO-MIN004RA) using MinKNOW v24.06.5.