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1.
Anticancer Res ; 41(9): 4609-4617, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34475089

RESUMO

BACKGROUND/AIM: This study aimed to assess the yield of an Oncomine comprehensive assay v3 (OCAv3)-based next-generation sequencing (NGS) analysis for detecting anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) fusions in non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: NGS data from 85 NSCLC cases were reviewed. ALK and ROS1 fusion status was compared to conventional tests. RESULTS: ALK or ROS1 fusion reads were detected in 17 NSCLC cases. Results in 10 NSCLC cases showed concordance with conventional tests, high-count fusion reads, a lack of mutually exclusive mutations of ALK or ROS1, and frequent signet-ring cell component. Seven NSCLC cases showing discordant results exhibited low to intermediate fusion read counts and mutations mutually exclusive from ALK or ROS1. CONCLUSION: Cases showing high-count fusion reads in OCAv3-based NGS have a strong possibility of carrying ALK or ROS1 fusion. Cases with low- to intermediate-count fusion reads should be interpreted with caution and may require additional confirmative tests.


Assuntos
Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Análise de Sequência de RNA/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Citogenética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa
2.
Nat Commun ; 12(1): 4825, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376658

RESUMO

Circular RNA (circRNA) is a class of covalently joined non-coding RNAs with functional roles in a wide variety of cellular processes. Their composition shows extensive overlap with exons found in linear mRNAs making it difficult to delineate their composition using short-read RNA sequencing, particularly for long and multi-exonic circRNAs. Here, we use long-read nanopore sequencing of nicked circRNAs (circNick-LRS) and characterize a total of 18,266 and 39,623 circRNAs in human and mouse brain, respectively. We further develop an approach for targeted long-read sequencing of a panel of circRNAs (circPanel-LRS), eliminating the need for prior circRNA enrichment and find >30 circRNA isoforms on average per targeted locus. Our data show that circRNAs exhibit a large number of splicing events such as novel exons, intron retention and microexons that preferentially occur in circRNAs. We propose that altered exon usage in circRNAs may reflect resistance to nonsense-mediated decay in the absence of translation.


Assuntos
Encéfalo/metabolismo , Éxons/genética , Íntrons/genética , Sequenciamento por Nanoporos/métodos , RNA Circular/genética , Análise de Sequência de RNA/métodos , Animais , Expressão Gênica , Humanos , Masculino , Camundongos da Linhagem 129 , Isoformas de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Methods Mol Biol ; 2351: 25-39, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382182

RESUMO

Post-transcriptional processing strongly affects the stability and the relative quantification of RNA molecules, so that steady-state levels of mature RNA, such as mRNAs, rarely reflect accurately the rate of in situ transcription in nuclei by RNA polymerases (RNAPs). The "Global Run-on Sequencing (GRO-Seq)" method, developed in 2008, combines the nuclear run-on assay with next-generation deep sequencing to detect nascent RNA levels to annotate the positions, the relative levels and the orientation of transcriptionally engaged RNA polymerase II (RNAPII) molecules genome-wide. Thus, GRO-Seq is a powerful method to infer mechanistic insights into the multiple levels of transcriptional regulation such as promoter-proximal pausing of RNAP, bidirectional transcription, and enhancer activity. Here, we describe a protocol for mammalian cells that can reliably detect low abundant nascent RNA from both coding and noncoding genomic regions. This protocol can easily be adapted for most mammalian cells to define the transcriptionally active regions of the genome and to measure dynamic transcriptional responses with high sensitivity upon external stimuli.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Polimerase II/metabolismo , Análise de Sequência de RNA/métodos , Transcrição Genética , Elementos Facilitadores Genéticos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Controle de Qualidade , RNA/genética , RNA/isolamento & purificação , RNA não Traduzido/genética
4.
Methods Mol Biol ; 2351: 41-65, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382183

RESUMO

Enhancers are transcribed by RNA polymerase II (Pol II). In order to study the regulation of enhancer transcription and its function in target gene control, methods are required that track genome transcription with high precision in vivo. Here, we provide step-by-step guidance for performing native elongating transcript sequencing (NET-Seq) in mammalian cells. NET-Seq allows quantitative measurements of transcription genome-wide, including enhancer transcription, with single-nucleotide and DNA strand resolution. The approach consists of capturing and efficiently converting the 3'-ends of the nascent RNA into a sequencing library followed by next-generation sequencing and computational data analysis. The protocol includes quality control measurements to monitor the success of the main steps. Following this protocol, a NET-Seq library is obtained within 5 days.


Assuntos
Elementos Facilitadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , Transcrição Genética , Animais , Células Cultivadas , Cromatina/genética , Biologia Computacional/métodos , DNA , Biblioteca Gênica , Humanos , Reação em Cadeia da Polimerase , RNA , RNA Polimerase II/metabolismo , Software
5.
Cancer Sci ; 112(9): 3555-3568, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34255396

RESUMO

The long reads of Nanopore sequencing permit accurate transcript assembly and ease in discovering novel transcripts with potentially important functions in cancers. The wide adoption of Nanopore sequencing for transcript quantification, however, is largely limited by high costs. To address this issue, we developed a bioinformatics software, NovelQuant, that can specifically quantify long-read-assembled novel transcripts with short-read sequencing data. Nanopore Direct RNA Sequencing was carried out on three hepatocellular carcinoma (HCC) patients' tumor, matched portal vein tumor thrombus, and peritumor to reconstruct the HCC transcriptome. Then, based on the reconstructed transcriptome, NovelQuant was applied on Illumina RNA sequencing data of 59 HCC patients' tumor and paired peritumor to quantify novel transcripts. Our further analysis revealed 361 novel transcripts dysregulated in HCC and that 101 of them were significantly associated with prognosis. There were 19 novel prognostic transcripts predicted to be long noncoding RNAs (lncRNAs), and some of them had regulatory targets that were reported to be associated with HCC. Additionally, 42 novel prognostic transcripts were predicted to be protein-coding mRNAs, and many of them could be involved in xenobiotic metabolism. Moreover, the tumor-suppressive roles of two representative novel prognostic transcripts, CDO1-novel (lncRNA) and CYP2A6-novel (protein-coding mRNA), were further functionally validated during HCC progression. Overall, the current study shows a possibility of combining long- and short-read sequencing to explore functionally important novel transcripts in HCC with accuracy and cost-efficiency, which expands the pool of molecular biomarkers that could enhance our understanding of the molecular mechanisms of HCC.


Assuntos
Carcinoma Hepatocelular/genética , Confiabilidade dos Dados , Neoplasias Hepáticas/genética , Sequenciamento por Nanoporos/métodos , Análise de Sequência de RNA/métodos , Transcriptoma , Idoso , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Biologia Computacional/métodos , Progressão da Doença , Feminino , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Software
7.
Commun Biol ; 4(1): 822, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193958

RESUMO

Stochastic gene expression leads to inherent variability in expression outcomes even in isogenic single-celled organisms grown in the same environment. The Drop-Seq technology facilitates transcriptomic studies of individual mammalian cells, and it has had transformative effects on the characterization of cell identity and function based on single-cell transcript counts. However, application of this technology to organisms with different cell size and morphology characteristics has been challenging. Here we present yeastDrop-Seq, a yeast-optimized platform for quantifying the number of distinct mRNA molecules in a cell-specific manner in individual yeast cells. Using yeastDrop-Seq, we measured the transcriptomic impact of the lifespan-extending compound mycophenolic acid and its epistatic agent guanine. Each treatment condition had a distinct transcriptomic footprint on isogenic yeast cells as indicated by distinct clustering with clear separations among the different groups. The yeastDrop-Seq platform facilitates transcriptomic profiling of yeast cells for basic science and biotechnology applications.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação Fúngica da Expressão Gênica/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Análise de Célula Única/métodos , Transcriptoma/genética , Análise por Conglomerados , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Guanina/metabolismo , Guanina/farmacologia , Ácido Micofenólico/metabolismo , Ácido Micofenólico/farmacologia , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/citologia , Análise de Sequência de RNA/métodos , Transcriptoma/efeitos dos fármacos
8.
Nat Commun ; 12(1): 4515, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34312385

RESUMO

The in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for 'reverse phenotyping'. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.


Assuntos
COVID-19/imunologia , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Linfócitos T/metabolismo , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , COVID-19/epidemiologia , COVID-19/virologia , Células Cultivadas , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , SARS-CoV-2/fisiologia , Linfócitos T/virologia
9.
BMC Plant Biol ; 21(1): 354, 2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315414

RESUMO

BACKGROUND: Atractylodes chinensis (DC.) Koidz is a well-known medicinal plant containing the major bioactive compound, atractylodin, a sesquiterpenoid. High-performance liquid chromatography (HPLC) analysis demonstrated that atractylodin was most abundant in 3-year old A. chinensis rhizome, compared with those from 1- and 2-year old rhizomes, however, the molecular mechanisms underlying accumulation of atractylodin in rhizomes are poorly understood. RESULTS: In this study, we characterized the transcriptomes from rhizomes of 1-, 2- and 3-year old (Y1, Y2 and Y3, respectively) A. chinensis, to identify differentially expressed genes (DEGs). We identified 240, 169 and 131 unigenes encoding the enzyme genes in the mevalonate (MVA), methylerythritol phosphate (MEP), sesquiterpenoid and triterpenoid biosynthetic pathways, respectively. To confirm the reliability of the RNA sequencing analysis, eleven key gene encoding factors involved in the sesquiterpenoid and triterpenoid biosynthetic pathway, as well as in pigment, amino acid, hormone and transcription factor functions, were selected for quantitative real time PCR (qRT-PCR) analysis. The results demonstrated similar expression patterns to those determined by RNA sequencing, with a Pearson's correlation coefficient of 0.9 between qRT-PCR and RNA-seq data. Differential gene expression analysis of rhizomes from different ages revealed 52 genes related to sesquiterpenoid and triterpenoid biosynthesis. Among these, seven DEGs were identified in Y1 vs Y2, Y1 vs Y3 and Y2 vs Y3, of which five encoded four key enzymes, squalene/phytoene synthase (SS), squalene-hopene cyclase (SHC), squalene epoxidase (SE) and dammarenediol II synthase (DS). These four enzymes directly related to squalene biosynthesis and subsequent catalytic action. To validate the result of these seven DEGs, qRT-PCR was performed and indicated most of them displayed lower relative expression in 3-year old rhizome, similar to transcriptomic analysis. CONCLUSION: The enzymes SS, SHC, SE and DS down-regulated expression in 3-year old rhizome. This data corresponded to the higher content of sesquiterpenoid in 3-year old rhizome, and confirmed by qRT-PCR. The results of comparative transcriptome analysis and identified key enzyme genes laid a solid foundation for investigation of production sesquiterpenoid in A. chinensis.


Assuntos
Atractylodes/metabolismo , Perfilação da Expressão Gênica/métodos , Transcriptoma/genética , Alquil e Aril Transferases/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Geranil-Geranildifosfato Geranil-Geraniltransferase/metabolismo , Transferases Intramoleculares/metabolismo , Análise de Sequência de RNA/métodos , Sesquiterpenos/metabolismo , Esqualeno Mono-Oxigenase/metabolismo
10.
Plant Sci ; 310: 110990, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34315604

RESUMO

Transfer cells (TCs) develop extensive wall ingrowths to facilitate enhanced rates of membrane transport. In Arabidopsis, TCs trans-differentiate from phloem parenchyma (PP) cells abutting the sieve element/companion cell complex in minor veins of foliar tissues and, based on anatomy and expression of SWEET sucrose uniporters, are assumed to play pivotal roles in phloem loading. While wall ingrowth deposition in PP TCs is a dynamic process responding to abiotic stresses such as high light and cold, the transcriptional control of PP TC development, including deposition of the wall ingrowths themselves, is not understood. PP TC development is a trait of vegetative phase change, potentially linking wall ingrowth deposition with floral induction. Transcript profiling by RNA-seq identified NAC056 and NAC018 (NARS1 and NARS2) as putative regulators of wall ingrowth deposition, while recent single cell RNA-seq analysis of leaf vasculature identified PP-specific expression of NAC056. Numerous membrane transporters, particularly of the UmamiT family of amino acid efflux carriers, were also identified. Collectively, these findings, and the recent discovery that wall ingrowth deposition is regulated by sucrose-dependent loading activity of these cells, provide new insights into the biology of PP TCs and their importance to phloem loading in Arabidopsis, establishing these cells as a key transport hub for phloem loading.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Floema/metabolismo , Proteínas de Arabidopsis/genética , Parede Celular/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Folhas de Planta/metabolismo , Análise de Sequência de RNA/métodos
11.
Methods Mol Biol ; 2314: 481-512, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235667

RESUMO

RNA sequencing (RNAseq) in bacteria has become a transformative tool for many applications, including the identification of mechanisms that contribute to pathogenesis, environmental adaptation, and drug response. The kinds of analysis outputs achievable from RNA-seq depend heavily on several key technical parameters during the sample preparation, sequencing, and data processing steps. In this chapter, we will describe the process of preparing Mycobacterium tuberculosis samples into sequencing libraries, selecting the appropriate sequencing platform, and performing data processing compatible with gene expression quantification. We will also discuss how each parameter could affect outcomes. The protocols described below produce consistently high yields. This chapter should inform on the technical considerations that impact sequencing output and enable the reader to decide on the best parameters to implement based on their own experimental goals.


Assuntos
Proteínas de Bactérias/genética , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Mycobacterium tuberculosis/genética , RNA Bacteriano/genética , Análise de Sequência de RNA/métodos , Humanos , RNA Bacteriano/análise , Fluxo de Trabalho
12.
Methods Mol Biol ; 2314: 513-531, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235668

RESUMO

Next-generation sequencing technologies facilitate the analysis of multiple important properties of transcriptomes in addition to gene expression levels. Here, we describe a method for mapping RNA 5' ends in Mycobacterium tuberculosis and Mycobacterium smegmatis, which allows the determination of transcription start sites (TSSs), comparative analysis of promoter usage under different conditions, and mapping of endoribonucleolytic cleavage sites. We describe in detail the procedures for constructing RNA sequencing libraries appropriate for RNA 5' end mapping using an Illumina sequencing platform, as well as bioinformatic procedures for data analysis.


Assuntos
Regiões 5' não Traduzidas/genética , Mycobacterium tuberculosis/genética , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , Análise de Sequência de RNA/métodos , Sítio de Iniciação de Transcrição , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Bacteriano/análise , Transcriptoma
13.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281207

RESUMO

miRNAs are involved in various biological processes, including adaptive responses to abiotic stress. To understand the role of miRNAs in the response to ABA, ABA-responsive miRNAs were identified by small RNA sequencing in wild-type Arabidopsis, as well as in abi1td, mkkk17, and mkkk18 mutants. We identified 10 novel miRNAs in WT after ABA treatment, while in abi1td, mkkk17, and mkkk18 mutants, three, seven, and nine known miRNAs, respectively, were differentially expressed after ABA treatment. One novel miRNA (miRn-8) was differentially expressed in the mkkk17 mutant. Potential target genes of the miRNA panel were identified using psRNATarget. Sequencing results were validated by quantitative RT-PCR of several known and novel miRNAs in all genotypes. Of the predicted targets of novel miRNAs, seven target genes of six novel miRNAs were further validated by 5' RLM-RACE. Gene ontology analyses showed the potential target genes of ABA-responsive known and novel miRNAs to be involved in diverse cellular processes in plants, including development and stomatal movement. These outcomes suggest that a number of the identified miRNAs have crucial roles in plant responses to environmental stress, as well as in plant development, and might have common regulatory roles in the core ABA signaling pathway.


Assuntos
Arabidopsis/genética , MicroRNAs/genética , Estresse Fisiológico/genética , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Genoma de Planta , MicroRNAs/metabolismo , Filogenia , Proteínas de Plantas/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , Transdução de Sinais/genética
14.
Medicine (Baltimore) ; 100(23): e25888, 2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34114985

RESUMO

BACKGROUND: Circular RNAs (circRNAs) play an important role in many neurological diseases and can serve as biomarkers for these diseases. However, the information about circRNAs in Parkinson disease (PD) remained limited. In this study, we aimed to determine the circRNAs expression profile in PD patients and discuss the significance of circRNAs in the diagnosis of PD. METHODS AND RESULTS: Using RNA-sequencing in peripheral blood RNAs, we showed that a significant number of mRNAs or circRNAs were differentially expressed between PD patients and normal controls (NCs), which included 273 up-regulated and 493 down-regulated mRNAs, and 129 up-regulated and 282 down-regulated circRNAs, respectively. Functional analysis was performed using the Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis, and the results showed that the second most enriched KEGG pathway was PD. These data suggest that the levels of mRNAs and circRNAs in peripheral blood could be potentially used as biomarkers for PD. In addition, we correlated mRNAs and circRNAs by constructing a competing endogenous RNA (ceRNA) network in PD. The resulted-in ceRNA network included 10 differentially expressed mRNAs from PD pathway, 13 predicted miRNAs, and 10 differentially expressed circRNAs. CONCLUSION: Collectively, we first characterized the expression profiles of circRNAs and mRNAs in peripheral blood from PD patients and proposed their possible characters in the pathogenesis of PD. These results provided valuable insights into the clues underlying the pathogenesis of PD.


Assuntos
Doença de Parkinson , RNA Circular , RNA Mensageiro , Análise de Sequência de RNA/métodos , Biomarcadores/sangue , China/epidemiologia , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Ontologia Genética , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação Oxidativa , Doença de Parkinson/sangue , Doença de Parkinson/diagnóstico , Doença de Parkinson/epidemiologia , Doença de Parkinson/genética , RNA Circular/sangue , RNA Circular/genética , RNA Mensageiro/sangue , RNA Mensageiro/genética
15.
J Hematol Oncol ; 14(1): 91, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108022

RESUMO

Single-cell sequencing, including genomics, transcriptomics, epigenomics, proteomics and metabolomics sequencing, is a powerful tool to decipher the cellular and molecular landscape at a single-cell resolution, unlike bulk sequencing, which provides averaged data. The use of single-cell sequencing in cancer research has revolutionized our understanding of the biological characteristics and dynamics within cancer lesions. In this review, we summarize emerging single-cell sequencing technologies and recent cancer research progress obtained by single-cell sequencing, including information related to the landscapes of malignant cells and immune cells, tumor heterogeneity, circulating tumor cells and the underlying mechanisms of tumor biological behaviors. Overall, the prospects of single-cell sequencing in facilitating diagnosis, targeted therapy and prognostic prediction among a spectrum of tumors are bright. In the near future, advances in single-cell sequencing will undoubtedly improve our understanding of the biological characteristics of tumors and highlight potential precise therapeutic targets for patients.


Assuntos
Genômica/métodos , Neoplasias/genética , Análise de Célula Única/métodos , Animais , Epigênese Genética , Perfilação da Expressão Gênica/métodos , Humanos , Neoplasias/patologia , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , Transcriptoma
16.
Methods Mol Biol ; 2348: 55-69, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34160799

RESUMO

RNA-sequencing could be nowadays considered the gold standard to study the coding and noncoding transcriptome. The great advantage of high-throughput sequencing in the characterization and quantification of long noncoding RNA (lncRNA) resides in its capability to capture the complexity of lncRNA transcripts configuration patterns, even in the presence of several alternative isoforms, with superior accuracy and discovery power compared to other technologies such as microarrays or PCR-based methods. In this chapter, we provide a protocol for lncRNA analysis using through high-throughput sequencing, indicating the main difficulties in the annotation pipeline and showing how an accurate evaluation of the procedure can help to minimize biased observations.


Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , RNA Longo não Codificante/genética , Análise de Sequência de RNA , Transcriptoma , Algoritmos , Interpretação Estatística de Dados , Bases de Dados Genéticas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Anotação de Sequência Molecular , Análise de Sequência de RNA/métodos , Software , Interface Usuário-Computador , Navegador
17.
Methods Mol Biol ; 2348: 71-90, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34160800

RESUMO

Mammalian genomes are pervasively transcribed and a small fraction of RNAs produced codify for proteins. The importance of noncoding RNAs for the maintenance of cell functions is well known (e.g., rRNAs, tRNAs), but only recently it was first demonstrated the involvement of microRNAs (miRNAs) in posttranscriptional regulation and then the activity of long noncoding RNAs (lncRNAs) in the regulation of miRNAs, DNA structure and protein function. LncRNAs have an expression more cell specific than other RNAs and basing on their subcellular localization exert different functions. In this book chapter we consider different protocols to evaluate the expression of lncRNAs at the single cell level using genome-wide approaches. We considered the skeletal muscle as example because the most abundant tissue in mammals involved in the regulation of metabolism and body movement. We firstly described how to isolate the smallest complete contractile system responsible for muscle metabolic and contractile traits (myofibers). We considered how to separate long and short RNAs to allow the sequencing of the full-length transcript using the SMART technique for the retrotranscription. Because of myofibers are multinucleated cells and because of it is better to perform single cell sequencing on fresh tissues we described the single-nucleus sequencing that can be applied to frozen tissues. The chapter concludes with a description of bioinformatics approaches to evaluate differential expression from single-cell or single-nucleus RNA sequencing.


Assuntos
Biologia Computacional/métodos , RNA Longo não Codificante/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Regulação da Expressão Gênica , Biblioteca Gênica , MicroRNAs/genética , Fibras Musculares Esqueléticas/metabolismo , Poliadenilação , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética
18.
Nat Methods ; 18(6): 604-617, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34099939

RESUMO

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.


Assuntos
Análise de Sequência de Proteína/métodos , Imagem Individual de Molécula/métodos , Espectrometria de Massas/métodos , Nanotecnologia , Proteínas/química , Proteômica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos
19.
Methods Mol Biol ; 2268: 21-42, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085259

RESUMO

A workflow is described for assaying the expression of G protein-coupled receptors (GPCRs) in cultured cells, using a combination of methods that assess GPCR mRNAs. Beginning from the isolation of cDNA and preparation of mRNA, we provide protocols for designing and testing qPCR primers, assaying mRNA expression using qPCR and high-throughput analysis of GPCR mRNA expression via TaqMan qPCR-based, GPCR-selective arrays. We also provide a workflow for analysis of expression from RNA-sequencing (RNA-seq) assays, which can be queried to yield expression of GPCRs and related genes in samples of interest, as well as to test changes in expression between groups, such as in cells treated with drugs or from healthy and diseased subjects. We place priority on optimized protocols that distinguish signal from noise, as GPCR mRNAs are typically present in low abundance, necessitating techniques that maximize sensitivity while minimizing noise. These methods may also be applicable for assessing the expression of members of families of other low abundance genes via high-throughput analyses of mRNAs, followed by independent confirmation and validation of results via qPCR.


Assuntos
Análise em Microsséries/métodos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Receptores Acoplados a Proteínas G/metabolismo , Análise de Sequência de RNA/métodos , Humanos , Cultura Primária de Células , RNA Mensageiro/genética , Receptores Acoplados a Proteínas G/genética
20.
Cell ; 184(13): 3573-3587.e29, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34062119

RESUMO

The simultaneous measurement of multiple modalities represents an exciting frontier for single-cell genomics and necessitates computational methods that can define cellular states based on multimodal data. Here, we introduce "weighted-nearest neighbor" analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of 211,000 human peripheral blood mononuclear cells (PBMCs) with panels extending to 228 antibodies to construct a multimodal reference atlas of the circulating immune system. Multimodal analysis substantially improves our ability to resolve cell states, allowing us to identify and validate previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets and to interpret immune responses to vaccination and coronavirus disease 2019 (COVID-19). Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets and to look beyond the transcriptome toward a unified and multimodal definition of cellular identity.


Assuntos
SARS-CoV-2/imunologia , Análise de Célula Única/métodos , Células 3T3 , Animais , COVID-19/imunologia , Linhagem Celular , Perfilação da Expressão Gênica/métodos , Humanos , Imunidade/imunologia , Leucócitos Mononucleares/imunologia , Linfócitos/imunologia , Camundongos , Análise de Sequência de RNA/métodos , Transcriptoma/imunologia , Vacinação
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