Chapter 22
RNA Sequencing for Transcript 5
0
-End Mapping
in Mycobacteria
M. Carla Martini, Huaming Sun, and Scarlet S. Shell
Abstract
Next-generation sequencing technologies facilitate the analysis of multiple important properties of transcriptomes in addition to gene expression levels. Here, we describe a method for mapping RNA 5
0 ends in
Mycobacterium tuberculosis and Mycobacterium smegmatis, which allows the determination of transcription
start sites (TSSs), comparative analysis of promoter usage under different conditions, and mapping of
endoribonucleolytic cleavage sites. We describe in detail the procedures for constructing RNA sequencing
libraries appropriate for RNA 5
0 end mapping using an Illumina sequencing platform, as well as bioinformatic procedures for data analysis.
Key words RNA-seq, RNA sequencing, RNA processing, RNase cleavage, Transcription start site,
RNA 5
0 ends, Endoribonuclease, RNase, Mycobacterium tuberculosis, Mycobacterium smegmatis
1 Introduction
RNA sequencing (RNA-seq) is widely used for comparative gene
expression analysis, but it is a flexible platform that allows for highresolution interrogation of a range of other transcriptomic features.
Transcript 5
0 ends are one such feature. They can be precisely
mapped by RNA-seq at single-nucleotide resolution, and the 5
0
ends produced by transcription initiation can be distinguished
from the 5
0 ends produced by ribonucleolytic cleavage. Since
these two types of transcript 5
0 ends represent entirely different
biological phenomena, distinguishing them is critical for understanding the biological basis of observed transcriptomic features.
Identification of transcription start sites (TSSs) allows for the direct
inference of promoter locations, facilitating analysis of transcriptional regulation. Identification of RNA cleavage sites provides
insight into RNA degradation pathways and posttranscriptional
regulation of gene expression.
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_22, © Springer Science+Business Media, LLC, part of Springer Nature 2021
513
RNA Sequencing for Transcript 5
0
-End Mapping
in Mycobacteria
M. Carla Martini, Huaming Sun, and Scarlet S. Shell
Abstract
Next-generation sequencing technologies facilitate the analysis of multiple important properties of transcriptomes in addition to gene expression levels. Here, we describe a method for mapping RNA 5
0 ends in
Mycobacterium tuberculosis and Mycobacterium smegmatis, which allows the determination of transcription
start sites (TSSs), comparative analysis of promoter usage under different conditions, and mapping of
endoribonucleolytic cleavage sites. We describe in detail the procedures for constructing RNA sequencing
libraries appropriate for RNA 5
0 end mapping using an Illumina sequencing platform, as well as bioinformatic procedures for data analysis.
Key words RNA-seq, RNA sequencing, RNA processing, RNase cleavage, Transcription start site,
RNA 5
0 ends, Endoribonuclease, RNase, Mycobacterium tuberculosis, Mycobacterium smegmatis
1 Introduction
RNA sequencing (RNA-seq) is widely used for comparative gene
expression analysis, but it is a flexible platform that allows for highresolution interrogation of a range of other transcriptomic features.
Transcript 5
0 ends are one such feature. They can be precisely
mapped by RNA-seq at single-nucleotide resolution, and the 5
0
ends produced by transcription initiation can be distinguished
from the 5
0 ends produced by ribonucleolytic cleavage. Since
these two types of transcript 5
0 ends represent entirely different
biological phenomena, distinguishing them is critical for understanding the biological basis of observed transcriptomic features.
Identification of transcription start sites (TSSs) allows for the direct
inference of promoter locations, facilitating analysis of transcriptional regulation. Identification of RNA cleavage sites provides
insight into RNA degradation pathways and posttranscriptional
regulation of gene expression.
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_22, © Springer Science+Business Media, LLC, part of Springer Nature 2021
513