RNA-Seq_Cp1002
Ontology highlight
ABSTRACT:
INSTRUMENT(S): AB SOLiD System 3.0
ORGANISM(S): Corynebacterium pseudotuberculosis
SUBMITTER: Rommel Ramos
PROVIDER: E-MTAB-2017 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Corynebacterium pseudotuberculosis strain 258, isolated from a caprine host in Brazil, was maintained in brain-heart-infusion broth (BHI HiMedia Laboratories Pvt. Ltda, India) at 37C, under rotation. Then, the bacteria was added to 20mL of BHI-Broth with 0.05% of 80% Tween and was maintained at 37C overnight, under rotation. The sample was then diluted 1/100 in 100mL of BHI broth, maintained at 37C, under rotation, and it was monitored until it reached the beginning of the exponential fase (A600 = 0.2). Afterwards, the sample was equally splitted in 4 aliquots of 20mL, centrifuged for 3 minutes under 8000rpm at 37C, and the pellets were re-suspendend in different medias according to the following conditions: acid stress, with HCl (fitting the pH to 5); osmotic stress, with 2M of NaCl; heat shock, where the pellet was re-suspended in pre-heated BHI (50C); and control, where the pellet was re-suspended in BHI under physiologic condition. Then, the samples for the acid stress, osmotic stress and control condition were maintained at 37C for 15 minutes, under rotation, whereas the sample under heat shock condition was maintained at 50C. Next, the samples were serially diluted from 10e-1 to 10e-6, where the samples from 10e-4 to 10e-6 were spread in BHI with agar (1.5%), which was maintained at 37C for 48hs for colony counting (duplicated samples); and the broth was centrifuged at 37C for 3 minutes under 8000rpm and the final pellet was re-suspended in 2mL of RNAlater, following the protocol of the manufacturer. Briefly, the bacteria suspended in RNAlater buffer were subjected to total RNA extraction using the ChargeSwitch total RNA cell kit (Invitrogen, USA) in accordance with the manufacturers recommendations, including the following adaptations: after the addition of the lysis buffer (Invitrogen), the material was transferred to 2mL tubes partially filled with 1mm diameter glass microbeads (Bertin Technologies). The cells were lysed mechanically using a Prescellys 24 homogenizer, set at 6,500 rpm, for 2 cycles (15 seconds per cycle) with an interval of 30 seconds between the cycles. The samples were centrifuged for 1 minute, and the supernatant transferred to fresh 2ml tubes and incubated in a dry bath at 60C for 15 minutes (representing the complete original protocol). DNAse was added to eliminate the residual genomic DNA. The elution of the total RNA from the magnetic beads was performed using 100 uL of milli-Q RNase-free water. The amount of total RNA was assessed using a Qubit 2.0 fluorometer (Invitrogen). In order to construct the library, the SOLiD Total RNA-Seq Kit was used. Briefly, after removing rRNAs, approximately 245ng of RNA was fragmented with RNAIII through incubation in a MJ Research thermocicler at 37C for 10 min and the reaction was interrupted with the addition of 90uL of nuclease-free water. The RNA was, then, concentrated through precipitation with ethanol according to the RiboMinus Concentration Module (Invitrogen - EUA), for subsequent hybridization with adapters and amplification of the cDNA library, which was obtained through reverse transcription using P1 adapters linked to the RNA extremities, according to the protocol SOLiD Total RNA-Seq Kit (Life Technologie, EUA). Next, the cDNA was denatured in polyacrylamide gel electrophoresis (6%) for obtaining fragments in the range from 150 to 250 base pairs for PCR amplification. The PCR amplifications were carried out in a MJ Research thermocycler as follow: the mixture was denatured at 95C for 30 sec, followed by 15 cycles of amplification at 95C for 30 sec, 62C for 30 sec and 72C for 30 sec, with a final extension of 72C for 7 min. Finally, the amplicons were purified using the kit PureLinkTM PCR Micro (Invitrogen - EUA), quantified in Qubit 2.0 Fluorometer-Invitrogen (EUA) and assessed through agarose electrophoresis 2%. The samples were fitted to the concentration of 60pg/mL, which is the suggested concentration for emulsion PCR. After the emulsion PCR, an enrichment reaction was performed for capturing only microbeads with fragments inserted. Next, a workflow analysis (WFA) was performed, where 15,000,000 microbeads were deposited to the slide and have undergone a binding cycle for quantitative and qualitative evaluation. According to this evaluation, 44,000,000 microbeads were deposited for sequencing, based on the manufacturer's description, generating reads of 50 nucleotides in range.
2020-06-20 | E-MTAB-9217 | biostudies-arrayexpress
Project description:MicroRNAs (miRNAs) are a class of small non-coding RNAs that function as post-transcriptional regulators of gene expression. Many miRNAs are expressed in the developing brain and regulate multiple aspects of neural development including neurogenesis, dendritogenesis and synapse formation. Rett syndrome (RTT) is a progressive neurodevelopmental disorder caused by mutations in the gene encoding Methyl-CpG binding protein 2 (MECP2). While Mecp2 is known to act as a global transcriptional regulator, miRNAs that are directly regulated by Mecp2 in the brain are not known. Using massively parallel sequencing methods, we have identified miRNAs whose expression is altered in cerebella of Mecp2-null mice before and after the onset of severe neurological symptoms. In vivo genome-wide analyses indicate that promoter regions of a significant fraction of dys-regulated miRNA transcripts, including a large polycistronic cluster of brain-specific miRNAs, are DNA methylated and directly bound by Mecp2. Functional analysis demonstrates that the 3’ untranslated region (UTR) of messenger RNA encoding Brain-derived neurotrophic factor (Bdnf) can be targeted by multiple miRNAs aberrantly up-regulated in absence of Mecp2. Taken together, these results suggest that dys-regulation of miRNAs may contribute to RTT pathoetiology, and also provide a valuable resource to further investigate the role of miRNAs in RTT. Two pooled total RNA samples (4 pairs of wild-type (WT) and Mecp2-null (KO) male mice; postnatal 6-week, the pre-/early-symptomatic stage) were sequenced in a multiplexed configuration (with distinct barcode sequences). And, six samples (two litters, one WT and two KO male mice in each litter; postnatal 8-week, the symptomatic stage) were sequenced individually.
2010-09-29 | E-GEOD-24320 | biostudies-arrayexpress
Project description:Rhabdomyosarcoma (RMS) is a highly malignant tumour accounting for nearly half of soft tissue sarcomas in children. Altered miRNA levels have been reported in human cancers, including RMS. Using deep sequencing technology, a total of 685 miRNAs were investigated in a group of alveolar RMSs (ARMSs), embryonal RMSs (ERMSs) as well as in normal skeletal muscle (NSM). Bioinformatics pipelines were used for miRNA target prediction and clustering analysis. Ninety-seven miRNAs were significantly deregulated in ARMS and ERMS when compared to NSM. MiR-378 family members were dramatically decreased in RMS tumour tissue and cell lines. Interestingly, members of the miR-378 family presented as a possible target the insulin-like growth factor receptor 1 (IGF1R), a key signalling molecule in RMS.
2015-01-28 | E-MTAB-3246 | biostudies-arrayexpress
Project description:To find potential microRNA links between Smad3 and E-cadherin, we characterized the microRNA profiles of two gastric cancer cell lines: SNU484-LPCX, which does not express Smad3, and SNU484-Smad3, in which Smad3 is overexpressed. We found that miR-200 families, among other differentially expressed miRNAs, are overexpressed in SNU484-Smad3. Through subsequent studies, including silencing of Smad3 in SNU484-Smad3 and expression profiling of epithelial-mesenchymal markers and ZEB1/2, known repressors of E-cadherin, we found that Smad3 regulates miR-200 families at the transcriptional level, which regulate ZEB 1/2, known transcriptional repressors of E-cadherin, at the post-transcriptional level. This represents an important link between the TGF-beta signaling pathway and post-transcriptional regulation by miRNAs. Examination of small RNA expression in 2 different cell lines (SNU484-LPCX, SNU484-Smad3).
2011-09-14 | E-GEOD-27977 | biostudies-arrayexpress
Project description:RNA sequencing (RNA-seq) on polyadenylated transcripts extracted from cardiomyocytes isolated from mice after 1 week of transverse aortic constriction (TAC) and in control mice (sham). 2-month-old male C57BL/6J mice were subjected under anesthesia to transverse aortic constriction for 1 week, then we performed RNA-seq on isolated cardiomyocytes
2013-11-30 | E-MTAB-1507 | biostudies-arrayexpress
Project description:We sought to investigate the extent of genetic and antigen-driven predetermination of antibody repertoires throughout B-cell development (pre-B cells, naive B cells, plasma cells). To this end, we used (i) an inbred model organism (mouse, C57BL/6J) to quantify genetic predetermination and (ii) cohort-stratification (4 cohorts: 1 untreated, 3 antigen-immunized) to quantify antigen-driven predetermination. To ensure generalizability of our research, we additionally sequenced naive B cells from Balb/c and pet shop mice.
2017-03-28 | E-MTAB-5349 | biostudies-arrayexpress
Project description:We generated the first recombinant strain library for fission yeast and conducted an RNA-seq based QTL study of the coding, non-coding, and antisense transcriptomes. We cross the lab strain 968 with the lab strain Y0036 to generate segregant. We used RNA-seq for both expression profiling and gentoyping of the segregant and DNA resequencing of the parental strains for variation detection in the whole library.
2014-12-04 | E-MTAB-2640 | biostudies-arrayexpress
Project description:Trans-splicing occurs post-transcriptionally and generates transcripts that are orderly inconsistent with their corresponding DNA templates. Until recently only exceedingly rare trans-splicing events have been experimentally characterized in the mammalian transcriptomes. Although hundreds to thousands of trans-spliced RNA candidates have been nominated by bioinformatics- or NGS (next-generation sequencing)-based approaches, these candidates unavoidably suffered from potential false positives arising from genetic rearrangement events or in vitro artifacts. Here we develop a pipeline (TSscan) based on NGS transcriptome data to identify trans-splicing in human embryonic stem cells (ESCs). TSscan integrates RNA sequencing data derived from different NGS platforms (i.e., Roche 454, SOLiD, and Illumina) and different human ESC lines (i.e., H1 and H9) as well as several in silico filters to minimize these two types of potential false positives. Our result shows that a tremendous amount of apparent experimental artifacts are indeed present in NGS data, which may be the most major false positives of trans-splicing detection. TSscan totally identified 10 trans-spliced RNA candidates in human ESCs, four of which are experimentally validated to be true. Further experiments reveal that these four events represent differential expression during the transition of pluripotent status to differentiate statuses. Especially, we observe that one event (the trans-spliced isoform of NCRMS), which is also a large intergenic non-coding RNA, tends to be specifically transcribed in ESCs and induced pluripotent stem cells and can conspicuously affect the pluripotency maintenance of ESCs. As far as we know, TSscan is the first pipeline for systematic identification of trans-splicing that utilizes NGS data in the human transcriptome, opening up an important class of post-transcriptional events for comprehensive characterization. human embryonic stem cell H9
2014-02-03 | E-GEOD-30557 | biostudies-arrayexpress
Project description:Background: Anaplasma phagocytophilum is an obligate intracellular prokaryotic pathogen that both infects and replicates within human neutrophils. The bacterium represses multiple antimicrobial functions while simultaneously increasing proinflammatory functions by reprogramming the neutrophil genome. Previous reports show that many observed phenotypic changes are in part explained by altered gene transcription. We recently identified that large chromosomal regions of the neutrophil genome are differentially expressed during A. phagocytophilum infection. Because of this, we sought to determine whether gene expression programs altered by infection were the result of changes in the host neutrophil DNA methylome. Results: Within 24 h of infection, marked increases in DNA methylation were observed genome-wide as compared with mock-infected controls and pharmacologic inhibition of DNA methyltransferases resulted in decreased bacterial growth. New regions of DNA methylation were enriched at intron and exon junctions; however, intragenic methylation did not correlate with altered gene expression. In contrast, intergenic DNA methylation was associated with A. phagocytophilum-induced gene expression changes. Within the major histocompatibility complex locus on chromosome 6, a region with marked changes in infection-induced differential gene expression, new regions of methylation were localized to boundaries of active and inactive chromatin. Conclusions: These data strongly suggest that A. phagocytophilum infection, in addition to altering histone structure, alters DNA methylation and the epigenome of its host cell to promote survival and replication, providing evidence that such bacterial infection can radically alter the epigenome of its host cell. Examination of methylated DNA sites in 3 human donors' neutrophils with and without 24h infection by Anaplasma phagocytophilum.
2015-09-22 | E-GEOD-73228 | biostudies-arrayexpress
Project description:Epithelial cells possess remarkable plasticity, having the ability to become mesenchymal cells through alterations in adhesion and motility (epithelial-to-mesenchymal transition or EMT). Recent studies suggest that EMT endows differentiated epithelial cells with stem cell traits, posing the interesting question of how epithelial plasticity is properly restricted to ensure epithelial differentiation during tissue morphogenesis. Here we identify zinc-finger transcription factor Ovol2 as a key suppressor of EMT of mammary epithelial cells. Epithelia-specific deletion of Ovol2 completely arrests mammary ductal morphogenesis, and depletes epithelial stem/progenitor cell reservoirs. Further, Ovol2-deficient epithelial cells undergo EMT in vivo to become non-epithelial cell types, and that Ovol2 directly represses key EMT inducers such as Zeb1 and regulates stem/progenitor cell responsiveness to TGF-beta. We also provide evidence for a suppressive role of Ovol2 in breast cancer progression. Our findings underscore the critical importance of exquisitely regulating epithelial plasticity to balance stemness with epithelial differentiation in development and cancer. We report ChIPseq data illustrating Ovol2 genome-wide targets in mouse mammary epithelial cells, suggesting that Ovol2 regulates a plethora of genes associated with the EMT process. Immunoprecipitated samples from HC11 mouse mammary epithelial cells with antibodies against Ovol2 and control IgG respectively were used for ChIP-seq experiments.
2014-03-19 | E-GEOD-53925 | biostudies-arrayexpress