RNA-seq of E. coli: E. coli MG1655 genome expressed in mRNA-optimized CFS (P10bff9)
The genome of Escherichia coli MG1655 was expressed in a cell-free system using the mRNA-optimized buffer (P10_Buffer 9) as described in the referenced study. Total RNA was extracted from 60 L of the cell-free reaction, purified via phenol/chloroform extraction, treated with DNase (Qiagen RNase-Free DNase Set), and cleaned using the Norgen RNA Clean-Up and Concentration Micro Kit. 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.