RESUMO
RNA-seq experiments are now routinely used for the large scale sequencing of transcripts. In bacteria or archaea, such deep sequencing experiments typically produce 10-50 million fragments that cover most of the genome, including intergenic regions. In this context, the precise delineation of the non-coding elements is challenging. Non-coding elements include untranslated regions (UTRs) of mRNAs, independent small RNA genes (sRNAs) and transcripts produced from the antisense strand of genes (asRNA). Here we present a computational pipeline (DETR'PROK: detection of ncRNAs in prokaryotes) based on the Galaxy framework that takes as input a mapping of deep sequencing reads and performs successive steps of clustering, comparison with existing annotation and identification of transcribed non-coding fragments classified into putative 5' UTRs, sRNAs and asRNAs. We provide a step-by-step description of the protocol using real-life example data sets from Vibrio splendidus and Escherichia coli.
Assuntos
Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Mensageiro/isolamento & purificação , RNA não Traduzido/isolamento & purificação , Archaea/genética , Bactérias/genética , Sequência de Bases , RNA Mensageiro/genética , RNA não Traduzido/genéticaRESUMO
The DRV technique (followed by extrusion) was used for construction of hydrophilic-USPIO encapsulating liposomes. Magnetoliposomes (ML) were characterized for size, surface charge, entrapment, physical stability and magnetic properties (relaxivity). Results show that nanosized extruded-DRV MLs encapsulate higher amounts of USPIOs in comparison with sonicated vesicles. Fe (III) encapsulation efficiency (EE) is 12%, the highest reported to date for nanosized MLs. EE of MLs is influenced by ML membrane composition and polyethyleneglycol (PEG) coating. PEG-coating increases ML EE and stability; however, r(2)-to-r(1) ratios decrease (in comparison with non-PEGylated MLs). Most ML-types are efficient T2 contrast agents (because r(2)-to-r(1) ratios are higher than that of free USPIOs). Targeted MLs were formed by successfully immobilizing OX-26 monoclonal antibody on ML surface (biotin-streptavidin ligation), without significant loss of USPIOs. Targeted MLs retained their nanosize and integrity during storage for 1 month at 4 °C and up to 2 weeks at 37 °C.