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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.
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.
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.
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:
5' overlap
)5'within_50bp
)annot.overlap
)3' overlap
)nearest.5'
)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.
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 |
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