RIViTseq enables systematic identification of regulons of transcriptional machineries I

Transcriptional regulation is a critical process to ensure expression of genes necessary for growth and survival in diverse environments. Transcription is mediated by multiple transcriptional factors including activators, repressors and sigma factors. Accurate computational prediction of the regulon of target genes for transcription factors is difficult and experimental identification is a laborious process and not scalable. Here, we demonstrate a new technology, RIViTseq, that enables systematic identification of regulons of transcription factors by combining an in vitro transcription assay and RNA-sequencing. Using this technology, target genes of 11 sigma factors were identified in Streptomyces coelicolor A3(2). The RIViTseq data expanded the transcriptional regulatory network in this bacterium, discovering new regulatory cascades and crosstalk between sigma factors. Implementation of RIViTseq with other transcription factors and in other organisms will improve our understanding of transcriptional regulatory networks across biology. RNA samples were prepared by incubating a mixture of a purified sigma factor, E. coli RNA polymerase core enzyme (NEB), digested genomic DNA and NTPs for 15 minutes at 30ºC. ERCC RNA Spike-in Mix was added as normalisation controls, genomic DNA was digested by DNase, and RNA samples were purified and sequenced on MiSeq System (Illumina) by paired-end 2 x 150 bp sequencing.