Combined transcriptome analysis and polysome profiling of Streptomyces coelicolor MT1110 in SMMS
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ABSTRACT: Total RNA, low ribosome density (LRD) and polysome associated RNA were obtained from parallel S.coelicolor cultures of wild type strain MT1110 grown on solid medium SMMS up to rapid growth phase II. The abundance of each RNA in the total RNA, the LRD or polysome pool was quantified by Cydye labeling and hybridization on high density whole genome 105K S.coelicolor DNA microarrays using S.coelicolor M145 as reference.
Project description:To obtain a separation of the epidermal and dermal compartments in order to examine compartment specific biological mechanisms in the skin we incubated 4 mm human skin punch biopsies in ammonium thiocyanate (NH4SCN). We wanted to test 1) the histological quality of the dermo-epidermal separation obtained by different incubation times 2) the amount and quality of extractable epidermal RNA, and 3) its impact on sample RNA expression profiles assessed by large-scale gene expression microarray analysis in both normal and inflamed skin. At 30 minutes incubation, the split between dermis and epidermis was not always histologically well-defined (i.e. occurred partly intra-epidermally) but varied between subjects. Consequently, curettage along the dermal surface of the biopsy was added to the procedure. This modified method resulted in an almost perfect separation of the epidermal and dermal compartments and satisfactory amounts of high-quality RNA were obtained. Hybridization to Affymetrix HG_U133A 2.0 GeneChips showed that ammonium thiocyanate incubation had a minute effect on gene expression resulting in only one significantly downregulated gene (cystatin E/M). We conclude that epidermis can be reproducibly and almost completely separated from the dermis of 4 mm skin biopsies by 30 min incubation in 3.8% ammonium thiocyanate combined with curettage of the dermal surface, producing high-quality RNA suitable for transcriptional analysis. Our refined method of dermo-epidermal separation will undoubtedly prove valuable in the many different settings, where the epidermal and dermal compartments need to be evaluated separately. Upper buttock skin in 4 healthy subjects was exposed to sodium lauryl sulphate, or sampled directly. For each subject, 4 biopsies were obtained: Two from inflamed skin, and two from adjacent normal skin. One irritated and one normal skin sample was placed directly in RNAlater. The remaining two samples were incubated in ammonium thiocyanate for 30 minutes at RT and then placed in RNAlater without performing any separation of the dermal and epidermal layers. This was done to investigate the effect of 30 minutes treatment with ammonium thiocyanate on both inflamed and non-inflamed skin. Data was normalized with quantile method (matrix 1) Forearm biopsies from 13 volunteers were separated to epidermis and dermis by use of ammonium thiocyanate. For comparison of full skin and epidermis without irritation, data from identical probe sets from HG_U133A 2.0 and HG_U133 plus 2.0 was extracted and normalised as one data set using quantile method (matrix 2).
Project description:Sexual reproduction is an ancient trait that evolved shortly after the appearance of the first eukaryotic cell. In order to study the transcriptional circuitries driving sexual reproduction in basidiomycota, we sequenced the poly(A)-positive transcriptome of stage 1 primordia and vegetative mycelia from the self-compatible dikaryotic basidiomycete Coprinopsis cinerea A43mutB43mut. Please note Okayama7 samples not included in this submission.
Project description:This SuperSeries is composed of the following subset Series: GSE31880: Resolution of ntla-dependent transcriptome at 9 hpf using caged molecules GSE31881: Resolution of ntla-dependent transcriptome at 16 hpf using caged molecules Refer to individual Series
Project description:Soil fungi are key players in biomass recycling. Predation influences fungal communities and modulates ecosystem services provided by fungi. Fungal chemical defense against predation comprises toxic proteins and secondary metabolites. The intent of this experiment was to generate transcriptomic information when a fungus, in this case Fusarium graminearum, was in the presence of a predator (Folsomia candida). We assumed that defense metabolites are synthesized on demand and transcriptome analysis can be used to pinpoint genes of defense pathways. To carry out the experiment, cultures of F. graminearum were subjected to grazing by springtail F. candida. After 48 hours at 15°C in dark, springtails were removed, and RNA was extracted from mycelium. Controls were incubated under the same conditions without animals. Each group consisted of four replicates. Strand-specific cDNA libraries were prepared using Illumina’s TruSeq stranded mRNA kit (75 bp paired-end) and sequenced on Illumina NextSeq 500V2.
Project description:Methyltransferase MRM2 methylates the 2’-hydroxyl group of ribonucleotide U1369 of human 16S mt-rRNA. Mutations in MRM2 have been implicated in human mitochondrial-related disease. This study investigates the role of MRM2 in the biogenesis and function of human mitochondrial ribosomes. Absence of MRM2 leads to a severe defect in mitochondrial translation, with the mtLSU being trapped in immature assembly states.
Project description:Mitochondrial translation was investigated by mitochondrial ribosome profiling (mitoRiboSeq) in three HEK293 cell lines: HEK293 wildtype, mtRF1 knockout 1, and mtRF1 knockout 2
Project description:Interested in how Adenovirus infection regulates temporal changes and differential export of viral and cellular mRNAs, we infected A549 cells with HAdV-C5 (H5pg4100) at 6, 12, 24 and 48 hours post-infection. Cells were infected with a MOI of 30 ffu and a non-infected (mock) sample was also included. After the infection period, cells were collected by trypsinization and fractionated to separate nucleus and cytoplasm with an NP-40 lysis buffer and RNA was extracted from each sample with TRIzol, followed by a column extraction using the RNeasy Plus Mini Kit. The cDNA libraries were made from mRNAs by first performing a poly(A) selection to the total RNA sample. The cDNA libraries were generated with the ScriptSeq v2 RNAseq Kit using their strand-specific protocol and a multiplex-sequencing was performed on the HiSeq 2500 platform using a paired-end run with a depth of approximately 50 million reads per sample. Two biological replicates of each infection time point and cellular fraction were sequenced using three technical replicates.
Project description:We have applied a recently developed, highly accurate and sensitive single-cell RNA-seq method (STRT/C1) to perform a molecular census of two regions of the mouse cerebral cortex: the somatosensory cortex and hippocampus CA1. We isolated cells fresh from somatosensory cortex (S1) and hippocampus CA1 area of juvenile (P22 - P32) CD1 mice, 33 males and 34 females. Cells were collected without selection, except that 116 cells were obtained by FACS from 5HT3a-BACEGFP transgenic mice. A total of 76 Fluidigm C1 runs were performed, each attempting 96 cell captures and resulting in 3005 high-quality single-cell cDNAs, containing Unique Molecular Identifiers allowing counting of individual mRNA molecules, even after PCR amplification.
Project description:A 10 ul drop of B. cinerea spores prepared in half-strength commercial grape juice was placed in the middle of detached Arabidopsis leaves. A cock-borer of 6 mm diameter was used to harvest leaf discs starting from the edge of the lesion (close, 0-6 mm) and the edge of the first disc (away, 6-12 mm) 48 hr after inoculation. Control plants were inoculated with a 10 ul drop of half-strength commercial grape juice with no spores. Three Biological replicates were done of which the third replicate was a dye-swap. In total 12 samples were analyzed. Each array constituted one replicate making six arrays for the whole experiment.