Ultra-deep RNA-seq of in vitro and in vivo transcripts by Escherichia coli RNA polymerase
We report a high-resolution Illumina RNA-seq method that can analyze non-coded base substitutions in mRNA at 10(-4)-10(-5) per base frequencies in vitro and in vivo. Overall design: The RNA samples were generated by transcription of pPR9 plasmid that contains a  5.7 kb fragment of E. coli rpoBC operon transcribed from a strong lambda phage PR promoter and terminated at an fd phage transcription terminator. The reference transcription reaction was performed in a buffer with 5 mM MgCl2 to determine the standard error rate (barcode 1). To reduce fidelity, we replaced Mg2+ with Mn2+ (barcode 2). To increase fidelity, we added GreA/GreB proteins for proofreading activity (barode 3 and 4). The same transcrit was purified from E. coli cells (barcode 5).