Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Dam-specific RNA-seq

ABSTRACT: To determine the transcription start site and the abundance of dam mRNA, we conducted mRNA-specific RNA-Seq experiments. Essentially two strategies were tested. First strategy: We obtained the cDNA with a specific RT-PCR in order to get the mRNA from our dam construct (see DNA-seq of DamID reporter cassette). An enrichment, in which we used a Biotinylated dam oligo and Streptavidin magnetic beads, was needed. Then, similar to SHAPE-seq, the ssDNA ligation was performed with CircLigase. The last step was the PCR reaction in order to prepare the cDNA libraries to be sequenced. Second strategy: Similar to the first one, but the specificity was introduced at the PCR, not at the RT. Thereby, busk cDNA was prepared with random hexamers. The enrichment step was not needed.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Mycoplasma pneumoniae

SUBMITTER: Eva Yus

PROVIDER: E-MTAB-5363 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants.

Yus Eva E Yang Jae-Seong JS Sogues Adrià A Serrano Luis L

Nature communications 20170828 1

Quantitative analysis of the sequence determinants of transcription and translation regulation is relevant for systems and synthetic biology. To identify these determinants, researchers have developed different methods of screening random libraries using fluorescent reporters or antibiotic resistance genes. Here, we have implemented a generic approach called ELM-seq (expression level monitoring by DNA methylation) that overcomes the technical limitations of such classic reporters. ELM-seq uses D ...[more]

PMID: 28848232

Similar Datasets

Project description:Background: Minimal change nephrotic syndrome (MCNS) is considered to be associated with T cell dysfunction, via unknown mechanisms. Experimental observations suggest that some humoral factors alter the permeability of glomerular filtration barrier. However, the nature of such factors remains still uncertain. Methods: Using cDNA microarrays, we performed gene expression profiling of peripheral blood mononuclear cells (PBMC) from three patients with MCNS during nephrosis and remission phases. To confirm the cDNA microarray results, we performed quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses in nephrosis and remission samples from 20 MCNS patients and six patients with nephrotic syndrome due to membranous nephropathy. Results: Out of 24,446 genes screened, 33 genes were up-regulated (at least 1.5-fold) in PBMC from these MCNS patients during the nephrosis phase. Up-regulated genes mainly encoded proteins involved in signal transduction and cytokine response. For further examination, we selected two genes encoding provable secretary proteins, chemokine (C-C) ligand 13 (also known as monocyte chemotactic protein-4) (CCL13) and a novel galectin-related protein (HSPC159). The results of RT-PCR showed that expressions of CCL13 and HSPC159 mRNA in nephrosis PBMC samples are higher than those in remission PBMC samples from all 20 MCNS patients examined. On the other hand, these mRNA expression patterns were variable among six patients with membranous nephropathy. Conclusions: We conclude that CCL13 and HSPC159 mRNA expressions in PBMC is up-regulated in MCNS patients during the nephrosis phase. These expression changes in PBMC might be involved in the pathophysiologic processes of MCNS. Using cDNA microarrays, we performed gene expression profiling of peripheral blood mononuclear cells (PBMC) from three patients with MCNS during nephrosis and remission phases. To confirm the cDNA microarray results, we performed quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses in nephrosis and remission samples from 20 MCNS patients and six patients with nephrotic syndrome due to membranous nephropathy.

Dataset Information

Dam-specific RNA-seq

Publications

A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure