|
| Status |
Public on Oct 15, 2016 |
| Title |
TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens. |
| Organisms |
Acinetobacter baumannii; Klebsiella aerogenes; Staphylococcus aureus; Staphylococcus epidermidis |
| Experiment type |
Expression profiling by high throughput sequencing
Other
|
| Summary |
Background
Bacteria rely on efficient gene regulatory mechanisms to switch between genetic programs when they are facing new environments. Although this regulation can occur at many different levels, one of the key steps is the initiation of transcription. Identification of the first nucleotide transcribed by the RNA polymerase is therefore essential to understand the underlying regulatory processes, since this provides insight on promoter strength and binding sites for transcriptional regulators, and additionally reveals the exact 5' untranslated region of the transcripts, which often contains elements that regulate translation.
Results
Here we present data from a novel TSS-EMOTE assay (Transcription Start Specific Exact Mapping Of Transcriptome Ends) to precisely map the transcription initiation sites of four entire transcriptomes. TSS-EMOTE is a variation of the dRNA-seq method, which has been combined with the EMOTE protocol, in order to increase detection of longer transcripts and limit biases introduced by PCR amplification of the Illumina sequencing library. Using TSS-EMOTE, 2018 promoters were detected in the opportunistic pathogen Staphylococcus aureus, and detailed consensus sequences could be obtained for the RNA polymerase recognition elements (e.g. sigma factor binding sites). The data also revealed a 94 nt median length of the 5' untranslated region in S. aureus, giving important insights for future work on translational regulation. Additionally, the transcriptomes of three other opportunistic pathogens, Staphylococcus epidermidis, Acinetobacter baumannii and Enterobacter aerogenes, were examined, and the identified promoter locations were then used to generate a map of the operon structure for each of the four organisms.
Conclusions
Mapping transcription start sites, and subsequent correlation with the genomic sequence, provides a multitude of important information about the regulation of gene expression, both at the transcriptional and translational level, by defining 5' untranslated regions and sigma-factor binding sites. We have here mapped transcription start sites in four important pathogens using TSS-EMOTE, a method that works with both long and 3'-phosphorylated RNA molecules, and which incorporates Unique Molecular Identifiers (UMIs) to allow unbiased quantification.
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| Overall design |
Each TSS-EMOTE assay consists of two library for the same sample: one library where RppH is added (+RppH) to allow ligation of the adapter at 5' end, one library without RppH added (-RppH) to establish the background signal. All TSS-EMOTE assays are performed in duplicate.
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| |
|
| Contributor(s) |
Prados J, Linder P, Redder P |
| Citation(s) |
27806702 |
| |
| Submission date |
Aug 02, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Julien Prados |
| E-mail(s) |
julien.prados@unige.ch
|
| Phone |
+41 22 37 95 396
|
| Organization name |
University of Geneva
|
| Department |
SCMU
|
| Lab |
Bioinformatics Support Platform
|
| Street address |
Rue Michel Servet 1
|
| City |
Geneva |
| ZIP/Postal code |
1211 |
| Country |
Switzerland |
| |
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| Platforms (4)
|
| GPL17452 |
Illumina HiSeq 2000 (Staphylococcus aureus) |
| GPL19644 |
Illumina HiSeq 2000 (Acinetobacter baumannii) |
| GPL21652 |
Illumina HiSeq 2000 (Staphylococcus epidermidis) |
| GPL22272 |
Illumina HiSeq 2000 ([Enterobacter] aerogenes) |
|
| Samples (30)
|
| GSM2257847 |
MW2 MH liquid 37C Bioreplicate1 without RppH |
| GSM2257848 |
MW2 MH liquid 37C Bioreplicate1 with RppH |
| GSM2257849 |
MW2 MH liquid 30C Bioreplicate1 without RppH |
| GSM2257850 |
MW2 MH liquid 30C Bioreplicate1 with RppH |
| GSM2257851 |
MW2 RPMI liquid 37C Bioreplicate1 without RppH |
| GSM2257852 |
MW2 RPMI liquid 37C Bioreplicate1 with RppH |
| GSM2257853 |
MW2 MH agar 37C Bioreplicate1 without RppH |
| GSM2257854 |
MW2 MH agar 37C Bioreplicate1 with RppH |
| GSM2257855 |
MW2 MH liquid 37C Bioreplicate2 without RppH |
| GSM2257856 |
MW2 MH liquid 37C Bioreplicate2 with RppH |
| GSM2257857 |
MW2 MH liquid 30C Bioreplicate2 without RppH |
| GSM2257858 |
MW2 MH liquid 30C Bioreplicate2 with RppH |
| GSM2257859 |
MW2 RPMI liquid 37C Bioreplicate2 without RppH |
| GSM2257860 |
MW2 RPMI liquid 37C Bioreplicate2 with RppH |
| GSM2257861 |
MW2 MH agar 37C Bioreplicate2 without RppH |
| GSM2257862 |
MW2 MH agar 37C Bioreplicate2 with RppH |
| GSM2257863 |
E. aerogenes Bioreplicate1 without RppH |
| GSM2257864 |
E. aerogenes Bioreplicate1 with RppH |
| GSM2257865 |
E. aerogenes Bioreplicate2 without RppH |
| GSM2257866 |
E. aerogenes Bioreplicate2 with RppH |
| GSM2257867 |
S. epidermidis Bioreplicate1 without RppH |
| GSM2257868 |
S. epidermidis Bioreplicate1 with RppH |
| GSM2257869 |
A. baumannii Bioreplicate1 without RppH |
| GSM2257870 |
A. baumannii Bioreplicate1 with RppH |
| GSM2257871 |
S. epidermidis Bioreplicate2 without RppH |
| GSM2257872 |
S. epidermidis Bioreplicate2 with RppH |
| GSM2257873 |
A. baumannii Bioreplicate2 without RppH |
| GSM2257874 |
A. baumannii Bioreplicate2 with RppH |
| GSM2257875 |
MW2 RPMI liquid 37C Bioreplicate1 mRNA-seq |
| GSM2257876 |
MW2 RPMI liquid 37C Bioreplicate2 mRNA-seq |
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| Relations |
| BioProject |
PRJNA336217 |
| SRA |
SRP080820 |
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