Unknown,Transcriptomics,Genomics,Proteomics

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Ultra-deep RNA-seq of in vitro and in vivo transcripts by Escherichia coli RNA polymerase


ABSTRACT: 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. 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).

ORGANISM(S): Escherichia coli

SUBMITTER: Masahiko Imashimizu 

PROVIDER: E-GEOD-46479 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Direct assessment of transcription fidelity by high-resolution RNA sequencing.

Imashimizu Masahiko M   Oshima Taku T   Lubkowska Lucyna L   Kashlev Mikhail M  

Nucleic acids research 20130807 19


Cancerous and aging cells have long been thought to be impacted by transcription errors that cause genetic and epigenetic changes. Until now, a lack of methodology for directly assessing such errors hindered evaluation of their impact to the cells. We report a high-resolution Illumina RNA-seq method that can assess noncoded base substitutions in mRNA at 10(-4)-10(-5) per base frequencies in vitro and in vivo. Statistically reliable detection of changes in transcription fidelity through ∼10(3) nt  ...[more]

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