Determining the transcriptome of an AdeAB deletion mutant in Acinetobacter baumannii S1
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ABSTRACT: RNA sequencing was carried out at BGI, Hong Kong on an Illumina HiSeq platform to compare gene expression in Acinetobacter baumannii strain S1 and an adeAB deletion mutant in this strain.
Project description:RNA sequencing was carried out by ARK genomics, Edinburgh on an Illumina HiSeq platform to compare gene expression in Acinetobacter baumannii strain AYE and an adeRS deletion mutant in this strain.
Project description:RNA sequencing was carried out at the University of Birmingham on an Illumina MiSeq platform to compare gene expression in Acinetobacter baumannii strain AYE and an adeB deletion mutant in this strain.
Project description:The aim of this experiment was to determine the changes in transcription caused by the loss of efflux function of AcrB. Three biological replicates per strain were grown in MOPS minimal medium to an OD600 of 0.6. Total RNA was extracted and DNase-treated. The samples were sent to BGI, Hong Kong for sequencing by Illumina Hiseq 4000.
Project description:The aim of this experiment was to determine the changes in transcription caused by inhibition of efflux pumps by the Phe-Arg-Beta-Naptylamide (PABN) and chlorpromazine. Four biological replicates per condition were grown to mid-log phase in MOPS minimal media (OD600) and were exposed to the known efflux inhibitor PABN, and the proposed efflux inhibitor chlorpromazine at 100 µg/ml and 50 µg/ml, respectively. Exposure lasted for two hours. Total RNA was extracted and DNase-treated. The samples were sent to BGI, Hong Kong for sequencing by Illumina Hiseq 4000.
Project description:Transcription factors are often regarded as being comprised of a DNA-binding domain and a functional domain. The two domains are considered separable and autonomous, with the DNA-binding domain directing the factor to its target genes and the functional domain imparting transcriptional regulation. We have examined a typical Zinc Finger (ZF) transcription factor from the Krüppel-like factor (KLF) family, KLF3. This factor has an N-terminal repression domain that binds the co-repressor C-terminal binding protein (CtBP), and a DNA-binding domain composed of three classical (ZFs) at its C-terminus. We established a system to compare the genomic occupancy profile of wildtype KLF3 with two mutants affecting the N-terminal functional domain: a mutant unable to contact its cofactor CtBP and a mutant lacking the entire N-terminal domain, but retaining the ZFs intact. We used chromatin immunoprecipitation followed by sequencing (ChIP-seq) to assess binding across the genome in murine embryonic fibroblasts. Our results define the in vivo recognition site for KLF3 and the two mutants as a typical CACCC-like element. Unexpectedly, we observe that mutations in the N-terminal functional domain severely affect DNA binding. In general, both mutations reduce binding but there are also instances where binding is retained or even increased. These results provide a clear demonstration that the correct localization of transcription factors to their target genes is not solely dependent on their DNA-contact domains. This informs our understanding of how transcription factors operate and is of relevance to the design of artificial ZF proteins. ChIP-seq was performed on the three samples, KLF3, ΔDL and DBD in duplicate (biological replicates). Input samples were used as controls.
Project description:We performed RNAseq for gene expression analysis for six strains of Acinetobacter Baumannii isolated from blood samples (defined as strains 1, 2, 3, 4 and 6) of patients hospitalized at the University Hospital \\"San Giovanni di Dio e Ruggi d'Aragona\\" (Salerno, Italy)
Project description:Using DNase-seq, mRNA-seq and publicly available ChIP-seq data sets, we examined the role of chromatin accessibility (DNase-seq) in androgen receptor binding to the genome (ChIP-seq) and AR-mediated transcriptional changes (mRNA-seq). Our data reveals genome-wide changes in chromatin structure that correspond to AR binding and differential gene expression. A focused examination of DNase-seq data around androgen receptor motifs within androgen receptor ChIP-seq peaks reveals distinct patterns of protection from DNaseI cleavage. Examination of chromatin accessibility (DNase-seq), AR binding (AR ChIP-seq), and transcription (mRNA-seq) in LNCaP cells before and after 12 hours of 1 nM R1881 treatment This Series represents the RNA-Seq data only. Exon microarray data generated under the same conditions is available through GSE15805. The DNase-seq data is publicly available through GSE32970 as well as the UCSC genome browser (genome.ucsc.edu) under Regulation::ENCODE DNase/FAIRE::Duke DNaseI HS:LNCaP and LNCaP + Andro. The accession numbers for the ChIP-seq experiments used are GSE14097 and GSE28126.
Project description:Polycistronic mRNAs transcribed from operons are resolved via the trans-splicing of a spliced leader (SL) RNA. The SL is also frequently trans-spliced to monocistronic transcripts. Using a modified cap analysis of gene expression (CAGE) protocol we mapped sites of SL trans-splicing genome-wide in the marine chordate Oikopleura dioica and find evidence for proposed functions of SL-trans-splicing. A recent hypothesis postulates that operons facilitate recovery from growth arrested states in metazoans. We examined the expression dynamics of operons across the life-cycle of the animal and during growth arrest recovery. We show that operons do not facilitate recovery from growth arrest in O. dioica. We find that operons are enriched in the germline and that trans-spliced transcripts are predominantly maternal., Interestingly, there is a TOP-like motif in the SL sequence, and trans-splicing in TOP mRNAs, indicating that trans-spliced mRNAs are targets for nutrient-dependent translational control in O. dioica. Total RNA from a number of stages across development were pooled and used in a modified DeepCAGE protocol. A custom designed spliced-leader primer (using the SL exon) was used in the 2nd strand synthesis step.
Project description:Background: A comprehensive transcriptome survey, or "gene atlas", provides information essential for a complete understanding of the genomic biology of an organism. We present an atlas of RNA abundance for 92 adult, juvenile and fetal cattle tissues and 3 cattle cell lines. Results: The Bovine Gene Atlas was generated from 7.2 million unique digital gene expression tag sequences (300.2 million total raw tag sequences), from which 1.59 million unique tag sequences were identified that mapped to the bovine genome accounting for 85% of the total raw tag abundance. Filtering these tags yielded 87,764 unique tag sequences that unambiguously mapped to 16,517 annotated protein-coding loci in the genome accounting for 45% of the total raw tag abundance. Clustering of tissues based on tag abundance profiles generally confirmed ontology classification based on anatomy. There were 5,429 constitutively expressed loci and 3,445 constitutively expressed unique tag sequences mapping outside annotated gene boundaries that represent a resource for enhancing current gene models. Physical measures such as inferred transcript length or antisense tag abundance identified tissues with atypical transcriptional tag profiles. We report for the first time the tissue specific variation in the proportion of mitochondrial transcriptional tag abundance. The Bovine Gene Atlas can be examined at http://www.agbase.msstate.edu/bovineatlas . Conclusions: The Bovine Gene Atlas is the deepest and broadest transcriptome survey of any livestock genome to date. Commonalities and variation in sense and antisense transcript tag profiles identified in different tissues facilitate the examination of the relationship between gene expression, tissue, and gene function. An atlas of mRNA abundance for 92 adult, juvenile and fetal cattle tissues and 3 cattle cell lines.
Project description:Genes that are constitutively expressed across multiple environmental stimuli are crucial to quantifying differentially expressed genes, particularly when employing quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assays. However, the identification of these potential reference genes in non-model organisms is challenging and is often guided by expression patterns in distantly related organisms. Here, transcriptome datasets from the diatom Thalassiosira pseudonana grown under replete, phosphorus-limited, iron-limited, and phosphorus and iron co-limited nutrient regimes were analyzed through literature-based searches for homologous reference genes, k-means clustering, and Analysis of Sequence Counts (ASC) to identify putative reference genes. A total of 9759 genes were identified and screened for stable expression. Literature-based searches surveyed 18 generally accepted reference genes, revealing 101 homologs in T. pseudonana with variable expression and a wide range of mean tags per million. K-means analysis parsed the whole transcriptome into 15 clusters. The two most stable clusters contained 709 genes but still had distinct patterns in expression. ASC analyses identified 179 genes that were stably expressed (posterior probability < 0.1 for 1.25 fold change). Genes known to have a stable expression pattern across the test treatments, like actin, were identified in this pool of 179 candidate genes. ASC can be employed on data without biological replicates and was more robust than the k-means approach in isolating genes with stable expression. The intersection of the genes identified through ASC with commonly used reference genes from the literature suggests that actin and ubiquitin ligase may be useful reference genes for T. pseudonana and potentially other diatoms. With the wealth of transcriptome sequence data becoming available, ASC can be easily applied to transcriptome datasets from other phytoplankton to identify reference genes. Axenic T. pseudonana CCMP 1335 was grown at 14°C under 24 hour light (120 µmol photons m-2 s-1) after Dyhrman et al. (2012) in f/2 plus silica chelated media made from surface Sargasso Sea water. Nitrate, silica, vitamins, and trace metals were at f/2 concentrations (Guillard and Ryther 1962), while iron and phosphate were modified across treatments. In brief, triplicate cultures of replete (36 µM PO4, 400 nM Fe), P-limited (0.4 µM PO4, 400 nM Fe), Fe-limited (36 µM PO4, 40 nM Fe), and Co-limited (0.4 µM PO4, 40 nM Fe) treatments were harvested when growth deviated from the replete control. Growth was monitored by cell counts. Biomass was harvested onto 0.2 µm filters and flash frozen in liquid nitrogen and total RNA was extracted as described in Dyhrman et al. (2012). Tag-seq sequencing of the transcriptome was performed by Illumina with a polyA selection and NlaIII digestion, resulting in 21 bp sequence reads or tags (Dyhrman et al., 2012). Libraries were of varied sizes as follows: replete (~12 million), P-limited (~13 million), Fe-limited (~23 million), and Co-limited (~26 million). Tags were mapped to gene models (predicted protein coding regions) with a pipeline designed by Genesifter Inc., requiring 100% identity and covering 9759 genes. Tag counts within a gene were pooled and normalized to the size of the library, with resulting data expressed in tags per million (tpm). Genes with normalized tag counts less than 2.5 tpm for each of the four treatments were excluded (Figure S1) , leaving 7380 genes in the analysis.