Project description:Detecting differential activation of transcription factors (TFs) in response to perturbation provides insight into global cellular processes. We present here Transcription Factor Enrichment Analysis (TFEA), a robust and reliable computational method that can detect differential activity of hundreds of TFs given any set of perturbation data. TFEA draws inspiration from GSEA and detects positional motif enrichment within an ordered list of regions of interest (ROI). As ROI are typically directly inferred from the data, we also introduce muMerge, a statistically principled method that generates a consensus list of ROIs from multiple replicates and conditions. TFEA is broadly applicable to data types that inform on transcriptional regulation, including CAGE data, ChIP-Seq, and accessibility data (e.g. ATAC-Seq). We demonstrate that TFEA can not only identify key TFs that respond to a perturbation, but also temporally unravel complex regulatory networks with time series data. Consequently, TFEA serves as a “hypothesis-generating engine” that provides an easy, rigorous, and cost-effective means to broadly assess TF activity to yield new biological insights about basic cellular processes.
2020-12-18 | GSE142419 | GEO
Project description:A Simplified Method to Enrich Tetrahymena Micronuclear DNA
Project description:Despite advances in RNA-seq, investigating the transcriptome of intracellular bacteria remains challenging due to the substantial presence of host RNA. In the case of Wolbachia spp. that are propagated in insect cell lines, commercially available rRNA depletion kits are often not suitable. Here, we describe a method to study the transcriptome of Wolbachia wAlbB in the Aedes albopictus cell line C6/36. To enrich the bacterial mRNA, eukaryotic mRNA was depleted by Dynabeads (Thermo Fisher Scientific). Then custom-designed riboPOOLs (siTOOLs Biotech) were used to remove both prokaryotic and eukaryotic rRNA. Compared to RNA prepared using the Illumina Ribo-Zero Plus rRNA Depletion Kit alone, additional depletion of eukaryotic mRNA increased wolbachial reads 7-fold to 0.7% of all reads. After removing eukaryotic and prokaryotic rRNAs with custom-designed riboPOOLs, there was a 300-fold increase of reads that mapped to Wolbachia (30.2%). Combining customized rRNA depletion from both organisms with eukaryotic mRNA depletion was more cost-effective than simply increasing the number of sequencing reads. This method can potentially be used for the enrichment of bacterial mRNA in studies of intracellular bacteria that cannot be propagated in standard cell lines.
Project description:Anthropogenic activities have dramatically increased the inputs of reactive nitrogen (N) into terrestrial ecosystems, with potentially important effects on the soil microbial community and consequently soil C and N dynamics. Our analysis of microbial communities in soils subjected to 14 years of 7 g N m-2 year-1 Ca(NO3)2 amendment in a Californian grassland showed that the taxonomic composition of bacterial communities, examined by 16S rRNA gene amplicon sequencing, was significantly altered by nitrate amendment, supporting the hypothesis that N amendment- induced increased nutrient availability, yielded more fast-growing bacterial taxa while reduced slow-growing bacterial taxa. Nitrate amendment significantly increased genes associated with labile C degradation (e.g. amyA and xylA) but had no effect or decreased the relative abundances of genes associated with degradation of more recalcitrant C (e.g. mannanase and chitinase), as shown by data from GeoChip targeting a wide variety of functional genes. The abundances of most N cycling genes remained unchanged or decreased except for increases in both the nifH gene (associated with N fixation), and the amoA gene (associated with nitrification) concurrent with increases of ammonia-oxidizing bacteria. Based on those observations, we propose a conceptual model to illustrate how changes of functional microbial communities may correspond to soil C and N accumulation.
Project description:A sigB null (∆sigB) mutant was constructed and analyzed for its phenotypes and transcriptome along with those of the parental RF122 strain. Method: Duplicate samples of rRNA depleted RNA from wild type and mutants were used to study transcriptomes by ion torrent platform.