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
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Shoman Mahmoud, Černý Martin, Jirát Matějčková Jitka, Schwarz Marek, Borah Nabajyoti, Vaňková Hausnerová Viola,
Neva Silvia, Šiková Michaela, Sanderova Hana, Halada Petr, Hubálek Martin, Dvořáková Věra, Převorovský Martin,
Holubová Jana, Staněk Ondřej, Krasny Libor, Zidek Lukas, Hnilicova Jarmila,
Expanding the CarD interaction network: CrsL is a novel transcription regulator in actinobacteria
Nucleic Acids Research, 2025; in press
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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:
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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
- Sikova et al:
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.
- Shoman 2025:
The sgRNA targeting crsL (MSMEG_5890) and carD (MSMEG_6077) were designed and cloned into
PLJR962 (for dCas9 expression) according to the previously established protocol1.
Mycobacterium smegmatis mc2 155 cells (negative control/NC, carD knockdown strain and crsL knockdown strain)
were inoculated to OD600 0.1 and grown at 37°C in Middlebrook 7H9 medium with 0.2% glycerol and 0.05% Tween 80.
For exponential [stationary] phase cells, knockdowns were induced with anhydrotetracycline (100 ng/mL)
after 3h [8h] of OD600 0.1 and the strains were cultivated for another 3h [16h] and harvested (OD600 ~0.5 [~2.5]).
The cells were pelleted by centrifugation and frozen at -70°C. For unbiased quantification,
RNA spike-in were added to each sample according to the following rule: 15 ng of
RNA spike-in were added per 30 ml culture of OD600~0.5.
1) Rock, J.M., et al., Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nature microbiology, 2017. 2(4): p. 1-9.
ChIPseq
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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), HelD (MSMEG_2174) and CrsL (MSMEG_5890) proteins.
All of these strains carried an extra copy of the genes encoding CarD, RbpA, HelD and CrsL 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.
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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.
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Mycobacterium smegmatis ΔHelD strain:
This experiment is complementary to the RNAP ChIP-seq in the wild type strain.
To see the influence of HelD on the distribution of RNA polymerase across the M. smegmatis chromosome,
we performed ChIP-seq experiment with the helD deletion strain. We used the antibody recognizing
RNA polymerase (the same one as in the ChIP-seq experiment with the wild type strain, see above)
and performed ChIP-seq in both exponential and stationary phase (see Kumar and Vankova 2024 for further details).
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:
- ORF starts within peak region (
5' overlap)
- ORF starts within +-50 bp peak region (
5'within_50bp)
- peak region is located within annotated region (
annot.overlap)
- ORF ends within peak region (
3' overlap)
- 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 |
| RNA-seq of Mycobacterium smegmatis mc2 155 negative control strain (NC), carD (MSMEG_6077) and crsL (MSMEG_5890) knockdown strains, from exponential and stationary phase cells. |
E-MTAB-13812 |
| 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 identification of CrsL (MSMEG_5890) binding sites in Mycobacterium smegmatis |
E-MTAB-12348 |
| ChIP-seq analysis of RNA polymerase and sigma A distribution in Mycobacterium smegmatis |
E-MTAB-12349 |
| ChIP-seq analysis of RNA polymerase and sigma A distribution in Mycobacterium smegmatis MSMEG_2174 (helD) deletion strain |
E-MTAB-13811 |
| RIP-seq identification of RNAs enriched on RNA polymerase and the primary sigma factor (sigma A) in Mycobacterium smegmatis |
E-MTAB-11692 |
Annotations
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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.
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The 'Mycobrowser' annotations for M. smegmatis were downloaded from
mycobrowser.epfl.ch release 4.
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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