Project description:Mesoplasma florum is a near-minimal bacterium belonging to the Mollicutes class particularly characterized by its small genome, fast growth rate, and lack of pathogenic potential. Here, we report the first genome-wide transcriptomic analysis of this microorganism using directional RNA sequencing (RNA-Seq) and 5'-rapid amplification of cDNA ends (5'-RACE). 5'-RACE sequencing reads were used to identify 605 putative transcription start sites (TSSs), of which more than 400 could be associated to a conserved promoter sequence. Along with terminator predictions, these TSSs allowed the reconstruction of M. florum transcription units, covering more than 90% of annotated genes. We evaluated the expression level of all M. florum protein coding genes using paired-end RNA-Seq reads according to the number of fragments per kilobase per million of mapped reads (FPKM). FPKM values were converted to absolute transcript abundances using biomass quantification data. These efforts aim at reaching a deep understanding of global cellular mechanisms in M. florum and will guide future genome engineering projects in this simple organism.
Project description:The near-minimal bacterium Mesoplasma florum constitutes an interesting model for synthetic genomics and systems biology studies due to its small genome, fast growth rate, and lack of pathogenic potential. However, some fundamental aspects of its biology remain largely unexplored. Here, we report a broad yet remarkably detailed characterization of M. florum by combining a wide variety of experimental approaches. More specifically, we investigated several physical and physiological aspects of this bacterium, and performed the first genome-wide analysis of its transcriptome and proteome using RNA sequencing techniques and two-dimensional liquid chromatography-tandem mass spectrometry. These analyses notably revealed the M. florum consensus promoter, a first experimental cartography of transcription units, as well as the transcription and expression levels of all annotated genes. We also converted gene transcription and expression levels into absolute molecular abundances using biomass quantification results, generating an unprecedented view of the M. florum cellular composition and functions. These characterization efforts will provide an experimental foundation for the development of a genome-scale metabolic model for M. florum and will guide future genome engineering endeavours in this simple organism.
Project description:We performed the RNA-seq in control samples and FXR1 knockdown samples, and compared the gene expression profiles to explore the effect of FXR1 knockdown on gene expression. The study was performed in H358 cells. Doxycycline inducible shRNA3 (sh3) was used to knockdown FXR1. Control shRNA (ctrl) samples were used to get rid of the effect of Doxycycline treatment. Both the Doxycycline treament for 3 days (D3) and 5 days (D5) samples were collected. Each sample has three repeats (rep 1, rep 2, and rep 3). The mRNA profiles were generated by deep sequencing using Illumina.Sequenced reads were trimmed for adaptor sequence, then mapped to hg19 whole genome using STAR v2.5.3 with parameters --bamRemoveDuplicatesType UniqueIdentical --outSAMmultNmax 1. Raw reads and Reads Per Kilobase per Megabase of library size (RPKM) were calculated using HOMER (PMID: 20513432). Differential gene expression was analyzed using R package DESeq2 using the raw reads.
