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De novo transcriptome profiling of highly purified human lymphocytes primary cells.

ABSTRACT: To help better understand the role of long noncoding RNAs in the human immune system, we recently generated a comprehensive RNA-seq data set using 63 RNA samples from 13 subsets of T (CD4(+)?naive, CD4(+)?TH1, CD4(+)?TH2, CD4(+)?TH17, CD4(+)?Treg, CD4(+)?TCM, CD4(+)?TEM, CD8(+)?TCM, CD8(+)?TEM, CD8(+)?naive) and B (B naive, B memory, B CD5(+)) lymphocytes. There were five biological replicates for each subset except for CD8(+)?TCM and B CD5(+)?populations that included 4 replicates. RNA-Seq data were generated by an Illumina HiScanSQ sequencer using the TruSeq v3 Cluster kit. 2.192 billion of paired-ends reads, 2×100?bp, were sequenced and after filtering a total of about 1.7 billion reads were mapped. Using different de novo transcriptome reconstruction techniques over 500 previously unknown lincRNAs were identified. The current data set could be exploited to drive the functional characterization of lincRNAs, identify novel genes and regulatory networks associated with specific cells subsets of the human immune system.

SUBMITTER: Bonnal RJ

PROVIDER: S-EPMC4587370 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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De novo transcriptome profiling of highly purified human lymphocytes primary cells.

Bonnal Raoul J P RJ Ranzani Valeria V Arrigoni Alberto A Curti Serena S Panzeri Ilaria I Gruarin Paola P Abrignani Sergio S Rossetti Grazisa G Pagani Massimiliano M

Scientific data 20150929

To help better understand the role of long noncoding RNAs in the human immune system, we recently generated a comprehensive RNA-seq data set using 63 RNA samples from 13 subsets of T (CD4(+) naive, CD4(+) TH1, CD4(+) TH2, CD4(+) TH17, CD4(+) Treg, CD4(+) TCM, CD4(+) TEM, CD8(+) TCM, CD8(+) TEM, CD8(+) naive) and B (B naive, B memory, B CD5(+)) lymphocytes. There were five biological replicates for each subset except for CD8(+) TCM and B CD5(+) populations that included 4 replicates. RNA-Seq data ...[more]

PMID: 26451251

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Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) infects many target cells (e.g., endothelial, epithelial, and B cells, keratinocytes, and monocytes) to establish lifelong latent infections. Viral latent-protein expression is critical in inducing and maintaining KSHV latency. Infected cells are programmed to retain the incoming viral genomes during primary infection. Immediately after infection, KSHV transcribes many lytic genes that modulate various cellular pathways to establish successful infection. Analysis of the virion particle showed that the virions contain viral mRNAs, microRNAs, and other noncoding RNAs that are transduced into the target cells during infection, but their biological functions are largely unknown. We performed a comprehensive analysis of the KSHV virion packaged transcripts and the profiles of viral genes transcribed after de novo infections of various cell types (human peripheral blood mononuclear cells [PBMCs], CD14(+) monocytes, and telomerase-immortalized vascular endothelial [TIVE] cells), from viral entry until latency establishment. A next-generation sequence analysis of the total transcriptome showed that several viral RNAs (polyadenylated nuclear RNA, open reading frame 58 [ORF58], ORF59, T0.7, and ORF17) were abundantly present in the KSHV virions and effectively transduced into the target cells. Analysis of the transcription profiles of each viral gene showed specific expression patterns in different cell lines, with the majority of the genes, other than latent genes, silencing after 24 h postinfection. We differentiated the actively transcribing genes from the virion-transduced transcripts using a nascent RNA capture approach (Click-iT chemistry), which identified transcription of a number of viral genes during primary infection. Treating the infected cells with phosphonoacetic acid (PAA) to block the activity of viral DNA polymerase confirmed the involvement of lytic DNA replication during primary infection. To further understand the role of DNA replication during primary infection, we performed de novo PBMC infections with a recombinant ORF59-deleted KSHV virus, which showed significantly reduced numbers of viral copies in the latently infected cells. In summary, the transduced KSHV RNAs as well as the actively transcribed genes control critical processes of early infection to establish KSHV latency.Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of multiple human malignancies in immunocompromised individuals. KSHV establishes a lifelong latency in the infected host, during which only a limited number of viral genes are expressed. However, a fraction of latently infected cells undergo spontaneous reactivation to produce virions that infect the surrounding cells. These newly infected cells are primed early to retain the incoming viral genome and induce cell growth. KSHV transcribes a variety of lytic proteins during de novo infections that modulate various cellular pathways to establish the latent infection. Interestingly, a large number of viral proteins and RNA are encapsidated in the infectious virions and transduced into the infected cells during a de novo infection. This study determined the kinetics of the viral gene expression during de novo KSHV infections and the functional role of the incoming viral transcripts in establishing latency.

Project description:BackgroundWatercress (Nasturtium officinale R. Br.) is an aquatic herb species that is a rich source of secondary metabolites such as glucosinolates. Among these glucosinolates, watercress contains high amounts of gluconasturtiin (2-phenethyl glucosinolate) and its hydrolysis product, 2-phennethyl isothiocyanate, which plays a role in suppressing tumor growth. However, the use of N. officinale as a source of herbal medicines is currently limited due to insufficient genomic and physiological information.ResultsTo acquire precise information on glucosinolate biosynthesis in N. officinale, we performed a comprehensive analysis of the transcriptome and metabolome of different organs of N. officinale. Transcriptome analysis of N. officinale seedlings yielded 69,570,892 raw reads. These reads were assembled into 69,635 transcripts, 64,876 of which were annotated to transcripts in public databases. On the basis of the functional annotation of N. officinale, we identified 33 candidate genes encoding enzymes related to glucosinolate biosynthetic pathways and analyzed the expression of these genes in the leaves, stems, roots, flowers, and seeds of N. officinale. The expression of NoMYB28 and NoMYB29, the main regulators of aliphatic glucosinolate biosynthesis, was highest in the stems, whereas the key regulators of indolic glucosinolate biosynthesis, such as NoDof1.1, NoMYB34, NoMYB51, and NoMYB122, were strongly expressed in the roots. Most glucosinolate biosynthetic genes were highly expressed in the flowers. HPLC analysis enabled us to detect eight glucosinolates in the different organs of N. officinale. Among these glucosinolates, the level of gluconasturtiin was considerably higher than any other glucosinolate in individual organs, and the amount of total glucosinolates was highest in the flower.ConclusionsThis study has enhanced our understanding of functional genomics of N. officinale, including the glucosinolate biosynthetic pathways of this plant. Ultimately, our data will be helpful for further research on watercress bio-engineering and better strategies for exploiting its anti-carcinogenic properties.

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De novo transcriptome profiling of highly purified human lymphocytes primary cells.

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De novo transcriptome profiling of highly purified human lymphocytes primary cells.

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