Two RNA-seq studies, one of coding and the other of coding and non-coding RNA for Identification of meningococcal factors required for colonization of epithelial and endothelial cells by high throughput screening
ABSTRACT: Two RNA-seq studies, one of coding and the other of coding and non-coding RNA for Identification of meningococcal factors required for colonization of epithelial and endothelial cells by high throughput screening
Project description:RNA-Seq analyses were performed in N. meningitidis strain Z5463 by deep sequencing using Ion Torrent technology (Thermo Fischer Scientific). For whole transcriptome libraries (mRNA+sRNA), cDNA libraries were prepared using the Ion Total RNA-Seq Kit v2 (Life Technologies) including a prior step of ribodepletion using specific probes adapted to N. meningitidis (Low Input Ribo Minus Eukaryote System v2) and size fractionation of RNA prior to cDNA synthesis with RNase III was performed. Briefly, 500 ng of total RNA were ribodepleted and then fragmented. After fragmentation, mean size of fragment was around 120 nt. Coding_and_NonCoding_R1, Coding_and_NonCoding_R2, and Coding_and_NonCoding_R3 represent three technical replicates. For sRNA libraries (sRNA), three cDNA libraries were prepared using the Ion Total RNA-Seq Kit v2 for Small RNA Libraries (Life Technologies) including a prior step of enrichment in small RNA fraction. Equal amounts of total RNA were used for the generation of all cDNA libraries. The three whole transcriptome libraries libraries (mRNA+sRNA) were then sequenced on an Ion ProtonTMSystem using the Ion PI Template and Sequencing OT 200 kit v3 and the 3 sRNA libraries were sequenced on an Ion PGM with the Hi-Q Template and Sequencing Kit using a 316 v2 array. NonCoding_R1, NonCoding_R2, and NonCoding_R3 represent three technical replicates.
Project description:In order to understand the role of long noncoding RNAs (lncRNAs) and their interaction with coding RNAs in esophageal sqaumous cell cancer (ESCC), we performed genome-wide screening of the expression of lncRNAs and coding RNAs from primary ESCC tissue and adjacent normal tissue using Agilent SurePrint G3 Human GE 8x60K Microarray. By comparing ESCC tissues and matched normal tissues, differentially expressed lncRNAs and coding RNAs were identified and confirmed with PCR and other independent studies. We further identified a subset of co-located and co-expressed lncRNAs and coding RNAs using bioinformatic tools and the analysis suggested that a subset of lncRNAs may influence nearby genes involved in the genesis of ESCC. Four pairs of ESCC primary tumors and adjacent normal tissues were used for genome-scale microarray experiments, which included long noncoding RNAs and coding RNAs. Selected lncRNAs expressed in the experiment were validated on independent matched-pair samples with PCR method.
Project description:Neisseria meningitidis is a human commensal that occasionally causes life-threatening infections such as bacterial meningitis and septicemia. Despite experimental evidence that gene regulation as well as the expression of small non-coding RNAs (sRNAs) affect meningococcal virulence, the organization of its transcriptome, including in particular the biogenesis of sRNAs and their mode of action, is only poorly understood. Here, we addressed these issues using a combination of high-throughput technologies. We applied differential RNA-seq (dRNA-seq) to produce a single-nucleotide resolution map of the primary transcriptome of N. meningitidis strain 8103. Our dRNA-seq analysis predicted 1,625 transcriptional start sites (TSS) including 65 non-coding RNA transcripts, of which 20 were further validated by Northern analysis. This allowed for the discovery of a novel CRISPR-associated sRNA with a Cas9-independent biogenesis. Genome-wide mapping of σ 70-dependent and independent promoters revealed that classical Escherichia coli-like σ70 promoter are absent in most of the protein coding genes in meningococci. The majority of the 706 primary TSSs (pTSSs) were associated with coding sequences, including 382 pTSS obtained for single genes and 240 pTSSs obtained for genes located in operons. By Hfq RNA immunoprecipitation sequencing (RIP-seq) we identified a large Hfq-centered post-transcriptional regulatory network comprising 24 sRNAs and 407 potential mRNA targets, and rifampicin stability assays demonstrated that Hfq binding confers enhanced stability on sRNAs. We finally confirmed the interactions of two sRNAs and their cognate target mRNA in vivo. Both directly repress prpB encoding a methylisocitrate lyse which was previously shown to be involved in meningococcal colonization of the human nasopharynx.The combination of both high-throughput approaches thus creates a compendium that not only provides a valuable data resource, but also allows for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx. Overall design: differential RNA-seq at two different growth phases; Hfq RIP-seq
Project description:Neisseria meningitidis is a commensal of humans that can colonize the nasopharyngeal epithelium for weeks to months and occasionally invades to cause life-threatening septicemia and meningitis. Comparatively little is known about meningococcal gene expression during colonization beyond those first few hours. In this study, the transcriptome of adherent serogroup B N. meningitidis strain MC58 was determined at intervals during prolonged cocultivation with confluent monolayers of the human respiratory epithelial cell line 16HBE14. At different time points up to 21 days, 7 to 14% of the meningococcal genome was found to be differentially regulated. The transcriptome of adherent meningococci obtained after 4 h of coculture was markedly different from that obtained after prolonged cocultivation (24 h, 96 h, and 21 days). Genes persistently upregulated during prolonged cocultivation included three genes (hfq, misR/phoP, and lrp) encoding global regulatory proteins. Many genes encoding known adhesins involved in epithelial adherence were upregulated, including those of a novel locus (spanning NMB0342 to NMB0348 [NMB0342-NMB0348]) encoding epithelial cell-adhesive function. Sixteen genes (including porA, porB, rmpM, and fbpA) encoding proteins previously identified by their immunoreactivity to sera from individuals colonized long term with serogroup B meningococci were also upregulated during prolonged cocultivation, indicating that our system models growth conditions in vivo during the commensal state. Surface-expressed proteins downregulated in the nasopharynx (and thus less subject to selection pressure) but upregulated in the bloodstream (and thus vulnerable to antibody-mediated bactericidal activity) should be interesting candidate vaccine antigens, and in this study, three new proteins fulfilling these criteria have been identified: NMB0497, NMB0866, and NMB1882. [Data is also available from http://bugs.sgul.ac.uk/E-BUGS-141]
Project description:To understand the role of long non-coding RNAs and interaction with coding RNAs in bladder urothelial cell carcinoma (BUCC), we performed genome-wide screening long non-coding RNAs and coding RNAs expression on primary BUCC tissues and normal tissues using long non-coding RNA array (Agilent plateform (GPL13825). By comparing these two groups, significantly differentially expressed lncRNAs and coding RNAs were identified. We further identifed a subset of long noncoding RNAs and their correlation with neighboring coding genes using bioinformatic tools. This analysis provides foundamental understaning of transcriptomic landscape changing during bladder carcinogenesis. 12 BUCC primary tumors and 3 normal tissues were used for long noncoding RNA array experiments which including long non-coding RNAs and coding RNAs. The differential expression of subset of long noncoding RNAs and their interaction with coding RNAs in BUCC compared with normal tissue will be identified with comtational analysis.
Project description:Expression was measured in 113 colorectal cancer samples using a costumized array. Expression was measured in 113 human colorectal cancer samples using a custom-designed array containing 26,910 non-coding RNA features and a set of 6,856 protein-coding genes.
Project description:Expression was measured in 113 colorectal cancer samples using a costumized array. Overall design: Expression was measured in 113 human colorectal cancer samples using a custom-designed array containing 26,910 non-coding RNA features and a set of 6,856 protein-coding genes.