MRNA degradation in Mycobacterium smegmatis under aerobic conditions
Ontology highlight
ABSTRACT:
ORGANISM(S): Mycolicibacterium smegmatis MC2 155
PROVIDER: GSE36342 | GEO | 2012/03/08
SECONDARY ACCESSION(S): PRJNA155559
REPOSITORIES: GEO
Similar Datasets
Project description:Transcription was arrested using 50 ug/ul of rifampin leaving RNA degradation as the sole vector responsible for mRNA abundance. mRNA half-lives were calculated from the decay of signal intensity from T0.
2012-03-08 | GSE36341 | GEO
Project description:Transcription was arrested using 50 ug/ul of rifampin leaving RNA degradation as the sole vector responsible for mRNA abundance. mRNA half-lives were calculated from the decay of signal intensity from T0. Three replicates were sampled before transcription arrest, and after 5 and 10 minutes of mRNA decay for a total of 9 arrays. Five minutes of RNA degradation occurred during the processing of these samples, therefore sample times, indicated in the sample name, represent 0, five, and ten minutes of RNA degradation.
2012-03-08 | E-GEOD-36342 | biostudies-arrayexpress
Project description:Transcription was arrested using 50 ug/ul of rifampin leaving RNA degradation as the sole vector responsible for mRNA abundance at 20C (cold) or in 0.2%O2 (hypoxia). mRNA half-lives were calculated from the decay of signal intensity from T0.
2012-03-08 | GSE36343 | GEO
Project description:Transcription was arrested using 50 ug/ul of rifampin leaving RNA degradation as the sole vector responsible for mRNA abundance. mRNA half-lives were calculated from the decay of signal intensity from T0. Four replicates were sampled after 0, 5 10, 15, 20, and 30 minutes of mRNA decay for a total of 24 arrays.
2012-03-08 | E-GEOD-36341 | biostudies-arrayexpress
Project description:After induction of DosR from a tet regulated promoter (uninduced controls were treated with an equivalent ammount of the solvent, DMSO), transcription was arrested using 50 ug/ul of rifampin leaving RNA degradation as the sole vector responsible for mRNA abundance. mRNA half-lives were calculated from the decay of signal intensity from T0.
2012-03-08 | GSE36344 | GEO
Project description:Transcription was arrested using 50 ug/ul of rifampin leaving RNA degradation as the sole vector responsible for mRNA abundance at 20C (cold) or in 0.2%O2 (hypoxia). mRNA half-lives were calculated from the decay of signal intensity from T0. Three replicates were sampled after 0, 1, 2, and 5 hours of mRNA decay in both cold and hypoxia for a total of 24 arrays.
2012-03-08 | E-GEOD-36343 | biostudies-arrayexpress
Project description:After induction of DosR from a tet regulated promoter (uninduced controls were treated with an equivalent ammount of the solvent, DMSO), transcription was arrested using 50 ug/ul of rifampin leaving RNA degradation as the sole vector responsible for mRNA abundance. mRNA half-lives were calculated from the decay of signal intensity from T0. Three replicates for each condition (DosR induced and uninduced) were sampled after 0, 10, 20, 30, and 60 minutes of mRNA decay for a total of 30 arrays.
2012-03-08 | E-GEOD-36344 | biostudies-arrayexpress
Project description:MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon macrophages. Infected and control (24 h incubation) macrophage mRNA samples were subjected to two rounds of amplification using the MessageAmp aRNA kit (Ambion) and manufacturer's instructions. Approximately 400 ng of macrophage mRNA was used as template in the first round of amplification, yielding 8.1 ug of infected macrophage aRNA and 7.8 ug of control macrophage aRNA. Approximately 1 ug of first-round macrophage aRNA was used as template in the second round of amplification, yielding 81.2 ug of infected macrophage aRNA and 83.3 ug of control macrophage aRNA. Infected and control macrophage aRNA samples were visualized on agarose gels to check quality and quantity. Except for the amounts of aRNA and reverse transcription (RT) primer used, macrophage target labeling reactions and microarray hybridizations were identical. Macrophage target syntheses used 5 ug of second-round aRNA and 5 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5'T18[Q-N]3'), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before. Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 72 Cy3, PMT 63 or 64 Cy5 for macrophage study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery). Keywords: other
2004-02-11 | GSE1012 | GEO
Project description:MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon head kidney. Head kidney mRNA samples were prepared from pooled (n = 10) infected or control kidney. Approximately 200 ng of infected or control head kidney mRNA was used as template in aRNA synthesis reactions. Two infected head kidney samples were pooled to yield 8.9 ug of aRNA, and a single control head kidney sample contained 8.4 ug of aRNA. Infected and control head kidney aRNA samples were visualized on agarose gels to check quality and quantity (data not shown). Except for the amounts of aRNA and reverse transcription (RT) primer used, head kidney target labeling reactions and microarray hybridizations were identical. Head kidney target syntheses used 2 ug of aRNA and 2 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C for 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5’T18[Q-N]3’), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before. Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 75 Cy3, PMT 63 or 64 Cy5 for head kidney study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery). Keywords: other
2004-02-11 | GSE1031 | GEO
Project description:The ability of Mycobacterium tuberculosis to infect, proliferate, and survive during long periods in the human lungs largely depends on the rigorous control of gene expression. Transcriptome-wide analyses are key to understanding gene regulation on a global scale. Here, we combine 5’-end-directed libraries with RNAseq expression libraries to gain insight into the transcriptome organization and post-transcriptional mRNA cleavage landscape in mycobacteria during log phase growth and under hypoxia, a physiologically relevant stress condition. Using the model organism Mycobacterium smegmatis, we identified 6,090 transcription start sites (TSSs) with high confidence during log phase growth, of which 67% were categorized as primary TSSs for annotated genes, and the remaining were classified as internal, antisense or orphan, according to their genomic context. Interestingly, over 25% of the RNA transcripts lack a leader sequence, and of the coding sequences that do have leaders, 53% lack a strong consensus Shine-Dalgarno site. This indicates that like M. tuberculosis, M. smegmatis can initiate translation through multiple mechanisms. Our approach also allowed us to identify over 3,000 RNA cleavage sites, which occur at a novel sequence motif. To our knowledge, this represents the first report of a transcriptome-wide RNA cleavage site map in mycobacteria. The cleavage sites show a positional bias toward mRNA regulatory regions, highlighting the importance of post-transcriptional regulation in gene expression. We show that in low oxygen, a condition associated with the host environment during infection, mycobacteria change their transcriptomic profiles and endonucleolytic RNA cleavage is markedly reduced, suggesting a mechanistic explanation for previous reports of increased mRNA half-lives in response to stress. In addition, a number of TSSs were triggered in hypoxia, 56 of which contain the binding motif for the sigma factor SigF in their promoter regions. This suggests that SigF makes direct contributions to transcriptomic remodeling in hypoxia-challenged mycobacteria. Taken together, our data provide a foundation for further study of both transcriptional and posttranscriptional regulation in mycobacteria.
2019-03-17 | GSE128412 | GEO