Details

This browser serves to display the data we collected by performing RNAseq, ChIPseq and RIPseq experiments in Mycobacterium smegmatis. Our goal was to identify novel RNA molecules bound to the transcription machinery and to map RNA polymerase and sigma factor position on the genome in both exponential and stationary phase of growth. All experiments were performed at 37°C in 7H9 medium supplemented with 10% glycerol and 0.05% Tween 80.

Related papers

• Viola Vaňková Hausnerová, Mahmoud Shoman, Dilip Kumar, Marek Schwarz, Martin Modrák, Jitka Jirát Matějčková, Eliška Mikesková, Silvia Neva, Anna Herrmannová, Michaela Šiková, Petr Halada, Iva Novotná, Petr Pajer, Leoš Shivaya Valášek, Martin Převorovský, Libor Krásný, Jarmila Hnilicová, RIP-seq reveals RNAs that interact with RNA polymerase and primary sigma factors in bacteria, Nucleic Acids Research, 2024;, gkae081, https://doi.org/10.1093/nar/gkae081

The displayed coverage data were computed as follows:

Reads were mapped with HISAT2, divided according to strand (when appropriate) and read coverage was computed with DeepTools bamComverage and scaled to 10^6 reads (additional scaling by factor of -1 for - strand). Finally, the shown mean coverage for corresponding sample groups was computed with wiggletools mean command.

RNAseq

The RNAseq data were taken from work by Šiková et. al (2019), ArrayExpress accession E-MTAB-7004
Šiková M, Janoušková M, Ramaniuk O, Páleníková P, Pospíšil J, Bartl P, et al. Ms1 RNA increases the amount of RNA polymerase in Mycobacterium smegmatis. Mol Microbiol. 2019;111(2):354-72. Epub 2018/12/11. doi: 10.1111/mmi.14159. PubMed PMID: 30427073.

ChIPseq

1. To reveal the distribution of mycobacterial transcription factors across the genome, we performed ChIPseq in the exponential phase of growth, using Mycobacterium smegmatis mc2 155 strains with FLAG tagged CarD (MSMEG_6077), RbpA (MSMEG_3858) and HelD (MSMEG_2174) proteins. All of these strains carried an extra copy of the genes encoding CarD, RbpA and HelD encoded by an integrative plasmid, whose expression was induced by the addition of anhydrotetracycline to the growth media. The proteins of interest with the respective genome fragments were isolated using anti-FLAG coated resin.
2. We used wild type Mycobacterium smegmatis mc2 155 cells for ChIPseq experiments using antibodies recognizing RNA polymerase and the main sigma factor Sigma A. In this series of experiments, we analyzed both exponential and stationary phase of growth.

Peak calling and gene assignment

The peak calling was done with MACS2 on each replicate separately and only the regions overlapping in all replicates were retained as resulting peaks. The highest (worst) p-value from the overlapping peaks was assigned to the resulting peak as its p-value.
The p-value and q-value for the peaks are reported as negative decadic logarithm of the respective statistics. The transparency of the peak changes linearly from 0 to 1 for 0 < -log(p-val) < 100 and is set to fully opaque for -log(p-val) > 100.

We assigned genes to each peak according to cascade of rules:

1. ORF starts within peak region (5' overlap)
2. ORF starts within +-50 bp peak region (5'within_50bp)
3. peak region is located within annotated region (annot.overlap)
4. ORF ends within peak region (3' overlap)
5. nearest ORF start (nearest.5')

RIPseq

To identify novel RNA molecules associating with transcription machinery, we performed RIPseq experiments (RNA immunoprecipitation followed by sequencing of isolated RNAs). We used wild type cells of Mycobacterium smegmatis mc2 155 in both exponential and stationary phase of growth. To precipitate RNA polymerase and the primary sigma factor sigma A, we used antibodies recognizing these proteins.

Source data

Sample ID	ArrayExpress accession
RNA-seq of wild type Mycobacterium smegmatis mc2 155 (exponential and stationary phase) and Ms1 deletion strain (exponential and stationary phase)	E-MTAB-7004
ChIP-seq identification of HelD (MSMEG_2174) binding sites in Mycobacterium smegmatis	E-MTAB-11827
ChIP-seq identification of CarD (MSMEG_6077) binding sites in Mycobacterium smegmatis	E-MTAB-11828
ChIP-seq identification of RbpA (MSMEG_3858) binding sites in Mycobacterium smegmatis	E-MTAB-11829
ChIP-seq analysis of RNA polymerase and sigma A distribution in Mycobacterium smegmatis	E-MTAB-12349
RIP-seq identification of RNAs enriched on RNA polymerase and the primary sigma factor (sigma A) in Mycobacterium smegmatis	E-MTAB-11692

Annotations

To make the 'NCBI' annotations visualization more consise in igv.js track representation, we have removed the "region" annotation spanning whole chromosome, added the "product" information from CDS to it's parrent gene and then removed the CDS annotation information.
Further, we've added the annotation for the Ms1 RNA (10.1093/nar/gku793, Rfam), to the NCBI annotation as gene-99999 with locus_tag MSMEG_Ms1.

The 'Mycobrowser' annotations for M. smegmatis were downloaded from mycobrowser.epfl.ch release 4.

The TSS data were obtained from work by Martini et. al (2019) Supplementary Table S2. Martini, M. C., Zhou, Y., Sun, H., & Shell, S. S. (2019). Defining the transcriptional and post-transcriptional landscapes of Mycobacterium smegmatis in aerobic growth and hypoxia. Frontiers in microbiology, 10, 591. doi: 10.3389/fmicb.2019.00591

References

• igv.js - embeddable genome browser github.com/igvteam/igv.js/, biorxiv.org/content/10.1101/2020.05.03.075499v1
• RefSeq - source of M. smegmatis sequence and 'NCBI' annotations 10.1093/nar/gkv1189
• Mycobrowser - source of M. smegmatis 'Mycobrowser' annotations 10.1016/j.tube.2010.09.006
• We thank w3schools.com for the w3.css stylesheet.

Download data

Files in the following directories contain processed data presented on this page. To obtain raw data, please use the ArrayExpress data repositories where the raw data for individual experiments are stored.

• RIPseq
• RNAseq
• ChIPseq