Bacterial Regulatory RNA (2^nd Ed., 2nd ed. 2024) Methods and Protocols Methods in Molecular Biology Series, Vol. 2741

This second edition details new and updated methods used for studying prokaryotic non-coding RNAs and their protein accomplices. Chapters detail discovery of ncRNAs, characterization of their structure, functions, and their interactomes. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Part I: sRNA discovery

1. RNA extraction from Gram-positive bacteria membrane vesicles using a polymer-based precipitation method

2. Extraction and purification of outer membrane vesicles and their associated RNAs

Anaïs Blache and Wafa Achouak

3. Analysis of phage regulatory RNAs: sequencing library construction from the fraction of small prokaryotic RNAs less than 50 nucleotides in length

Sylwia Bloch, Natalia Lewandowska, Wojciech Wesołowski, Aleksandra Łukasiak, Paulina Mach, Bożena Nejman-Faleńczyk and Grzegorz Węgrzyn.

4. Discovering Novel Bacterial Small RNA by RNA-seq Analysis Toolkit ANNOgesic

Chin-Hsien Tai, Deborah Hinton, and Sung-Huan Yu

Part II: sRNA functions

Maximilian P. Kohl, Béatrice Chane-Woon-Ming, Roberto Bahena-Ceron, José Jaramillo, Laura Antoine, Lucas Herrgott, Pascale Romby, and Stefano Marzi

Gianluca Prezza andAlexander Westermann

7. Investigation of sRNA-mRNA interactions in Bacillus subtilis in vivo

Inam Ul Haq, Peter Müller, and Sabine Brantl

8. In vitro methods for the investigation of sRNA-mRNA interactions in Bacillus subtilis

9. RNA double helix hybridization measured by fluorescence correlation spectroscopy

Arne Werner

10. New perspectives on cross talks between bacterial regulatory RNAs from outer membrane vesicles and eukaryotic cells

Moumita Roy Chowdhury and Eric Massé

11. Experimental validation of RNA-RNA interactions by Electrophoretic Mobility Shift Assay

Eva Maria Sternkopf Lillebæk and Birgitte Haahr Kallipolitis.

12. Dynamics and function of sRNA/mRNAs under the scrutiny of computational simulation methods

Agustín Ormazábal, Juliana Palma, and Gustavo Pierdominici-Sottile

Robina Scheuer, Jennifer Kothe, Jan Wähling, and Elena Evguenieva-Hackenberg

14. Evaluation of 5'-end phosphorylation for small RNA stability and target regulation in vivo.

Alexandra Schilder, Yvonne Göpel, Muna Ayesha Khan, and Boris Görke

15. In-gel cyanoethylation for pseudouridines mass spectrometry detection of bacterial regulatory RNA.

Antony Lechner and Philippe Wolff

Part III: sRNA interactome

16. Directed screening for sRNA targets in E. coli using a plasmid library.

Xing Luo and Nadim Majdalani

17. Defining Bacterial RNA-RNA Interactomes Using CLASH

Sofia Esteban-Serna, Liang-Cui Chu, Mehak Chauhan, Pujitha Raja, an dSander Granneman

Thomas Søndergaard Stenum and Erik Holmqvist

19. An integrated affinity chromatography-based approach to unravel the sRNA interactome in nitrogen-fixing rhizobia

Natalia Isabel García-Tomsig, Antonio Lagares Jr., Anke Becker, Claudio Valverde, and José Ignacio Jiménez-Zurdo

Part IV: sRNA structure

20. sRNA structural modeling based on NMR data

Pengzhi Wu and Lingna Yang

21. Circular and linear dichroism for the analysis of small noncoding RNA properties

Florian Turbant, Kevin Mosca, Florent Busi, Véronique Arluison, and Frank Wien

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts