Whole-genome Identification of Transcriptional Start Sites by Differential RNA-seq in Bacteria

Abstract

Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene structure, we are able to identify two kinds of transcriptional start site: primary and secondary. A primary transcriptional start site is located some nucleotides upstream of the translational start site, while a secondary transcriptional start site is located within the gene encoding sequence. Here, we present a step by step protocol for genome-wide transcriptional start sites determination by differential RNA-sequencing (dRNA-seq) using the enteric pathogen Shigella flexneri serotype 5a strain M90T as model. However, this method can be employed in any other bacterial species of choice. In the first steps, total RNA is purified from bacterial cultures using the hot phenol method. Ribosomal RNA (rRNA) is specifically depleted via hybridization probes using a commercial kit. A 5'-monophosphate-dependent exonuclease (TEX)-treated RNA library enriched in primary transcripts is then prepared for comparison with a library that has not undergone TEX-treatment, followed by ligation of an RNA linker adaptor of known sequence allowing the determination of TSS with single nucleotide precision. Finally, the RNA is processed for Illumina sequencing library preparation and sequenced as purchased service. TSS are identified by in-house bioinformatic analysis. Our protocol is cost-effective as it minimizes the use of commercial kits and employs freely available software.

Keywords: 5′-monophosphate-dependent exonuclease (TEX); Bacterial gene regulation; Hot phenol RNA extraction; Phenol chloroform:isoamyl alcohol RNA extraction; RNA phosphorylation; RNA precipitation; RNA purification; TSS; Transcriptional start site; dRNA-seq; rRNA depletion.

Figure 1.. Schematic representation of the Primary and Secondary Transcriptional Start Site definition

Figure 2.. Workflow of dRNA-seq for whole-genome Transcriptional Start Sites identification

Figure 3.. Total RNA quality control.

A. Separation of total RNA from three replicates in a 1% agarose gel in TAE of Shigella flexneri 5a M90T as total RNA quality control. B. Examples of typical values obtained from total RNA quantification in a NanoDrop. The 260/280 ratio should be around 1.88-2.00.

Figure 4.. Total RNA quality control after DNase treatment.

A. Separation of total RNA from three replicates in a 1% agarose gel in TAE of Shigella flexneri 5a M90T as total RNA quality control. B. Examples of typical values obtained from total RNA quantification after DNase treatment in a NanoDrop.

Figure 5.. Control PCR to verify the absence of genomic DNA with the hfq gene plus 300 pb upstream and downstream of Shigella flexneri 5a M90T as target using genomic DNA as a positive control.

Agarose gel 1% in TAE 1x. The absence of a PCR product seen as a 909 bp band in lanes 2-4 indicates that the purified RNA is devoid of DNA contaminations.

Figure 6.. Shigella flexneri 5a M90T RNA treated (TEX+) and untreated (TEX-) with TEX

Figure 7.. FastQC report of quality control.

A. Successful quality control. The quality scores should be in the green area for the entire length of the sequenced fragment. B. Unsuccessful quality control. The quality scores for part of the reads are low (found in the orange or red areas).

Figure 8.. Screenshot of the IGV browser for alignment visualization.

Alignment of TEX+ and TEX- libraries with the reference genome of S. flexneri 5a M90T. The blue histogram shows the coverage of the TEX- sample, the red histogram shows the TEX+ sample coverage. The red highlight shows the putative transcriptional start site.

Figure 9.. Example of extract from the ReadXplorer output file