Reconhecemos o apoio da Ciência Química, Geociências e Biociências Divisão, Escritório de ciências básicas da energia, Secretaria de Ciência, Departamento de Energia dos EUA (número prêmio DE-FG02-91ER20021) e National Science Foundation (MCB 0.948.584) (FB). Somos gratos à Sra. Karen Pássaro para a edição do manuscrito.

1. EMS Tratamento Sementes de Arabidopsis thaliana são mutagenizado utilizando como agente mutagénico sulfonato de metano agente de etilo (EMS) 3,4, o que induz nos C-para-T genoma alterações resultantes em C / G para T / A mutações 5-7. <ol><li> Pesar 0,8 g de sementes de Arabidopsis (~ de 40.000 sementes) que transportam a organela marcador fluorescente (especificamente, neste estudo ssGFPHDEL (sequência sinal-GFP-HDEL tetrapéptido) tem sido utilizado como um marcador de ER). </li><li> Transferir as sementes em um tubo Falcon de 50 ml, em seguida, adicionar 25 ml de água destilada. </li><li> Adicionar 0,2% (v / v) etil-sulfonato de metano. </li><li> Incubar durante 16 horas em nutação misturador a baixa velocidade. </li><li> Aspirar o líquido e descartá-lo para um frasco contendo 1,0 M de NaOH para inactivar a EMS. </li><li> Adicionar 25 ml de água para o tubo Falcon contendo as sementes, fechar e invertido 5 vezes para lavar as sementes; esperar até que todas as sementes se instalaram, em seguida, aspire a água e descarte-o a 1,0 M Na<html> OH balão. </li><li> Repetir o passo de lavagem até 10 vezes. </li><li> Após a lavagem final, re-suspender as sementes numa quantidade mínima de água. </li><li> Proceder à esterilização de sementes adicionando 25 mL de água sanitária a 10%, agitar vigorosamente por 30 s. Vamos resolver sementes para baixo, em seguida, deitar fora lixívia e lavar com 25 mL de água estéril. Decantar a água estéril e adicionar 25 mL de etanol a 70%. Agitar os tubos por 30 s. Vamos resolver sementes para baixo. Decantar o etanol e lavar com 25 mL de água estéril. Repetir duas vezes a lavagem com água esterilizada, em seguida, deitar sementes em uma placa de Petri de plástico contendo 3 MM de papel filtro e deixe secar sob o capô. Armazenar a 4 ° C durante 2 dias. </li><li> Placa de as sementes em ½ MS phytagel (concentração metade do meio de Murashige e Skoog), 150-milímetros placa de Petri (aprox. 250-300 sementes para prato). </li><li> Crescer sementes M1 na placa para 2 semanas, depois transplantar para o solo. </li><li> Coletar sementes M2 de plantas individuais M1 para gerar linhas de M2 ​​(1000 linhas independentes). </li></ol>ove_title &quot;&gt; 2 Triagem. Confocal de Populações M2 e M3 Nesta secção descreve-se a observação de plântulas com um microscópio confocal de fluorescência ou como descrito anteriormente 8. <ol><li> Sessenta sementes de cada linha M2 são cultivadas durante 7 a 10 dias, em pratos de plástico de Petri, ½ MS Phytagel; na mesma placa também são cultivadas 5 mudas EMS-não tratado (controlo). </li><li> Cinco a 10 cotilédones são montados com o lado abaxial para a lente (40X) numa lâmina de microscópio e fechado com uma lamela. </li><li> Cada cotilédone é observado, a partir do cortical para a região medial, sob fluorescência para qualquer distribuição alterada subcelular do marcador organela. </li><li> Plantas positivas são transplantadas para o solo e as suas sementes são recolhidas e peneirada novamente para confirmar o fenótipo mutante na geração M3. </li><li> Remover os contaminantes mutações de fundo através de retrocruzamento, pelo menos, três vezes para um genoma de tipo selvagem, possivelmente, containing o desejado marcador organela fluorescente. <ol><li> A partir da planta-mãe, usando tesouras ou pinças finas remover siliques maduros e flores abertas. </li><li> Remova os botões que são demasiado pequenos do meristema. </li><li> Insira a ponta de um par de fórceps entre pétalas e sépalas de abrir um botão de flor e remover todas as anteras. </li><li> Usando um par de fórceps levar uma flor aberta maduro da planta pai e esfregar as anteras sobre o estigma da planta emasculado. </li></ol></li></ol> 3. Mapeamento Esta secção descreve essencialmente como mapear uma mutação recessiva usando um protocolo modificado de Borevitz 9, que é mais rápido se comparado com os métodos tradicionais de mapeamento 10,11. Esta abordagem utilizar matrizes de alta densidade de oligonucleótidos com a capacidade de detectar polimorfismos numerosas única característica (SFPs) num único ensaio 12. Usando o Arabidopsis Affymetrix matriz GeneChip ATH1 é possívelanalisar aproximadamente 24.000 genes. Um conjunto de indivíduos F 2 mostrando a mutação é comparado a um pool de tipo selvagem de plantas F 2 recolhidos dentro da mesma população de segregar. Em seguida, a mutação irão ser mapeado na região onde o pool mutante é enriquecido para alelos de genótipos mutantes e, consequentemente, na mesma região da piscina do tipo selvagem irá resultar enriquecida para os alelos de tipo selvagem-mãe 13. <ol><li> O ADN genómico (3 ug) é extraído do homozigótica mutante Columbia (M3), utilizando um Qiagen DNeasy Plant Kit Mini e é submetido à Illumina Genome Analyzer II (GA II) 14 sequenciação. </li><li> O mutante homozigótica Columbia (M3) é cruzada com Landsberg erecta para gerar uma população de mapeamento. </li><li> O mapeamento é realizado em 70 até 100 indivíduos F2 mostrando o fenótipo aberrante e do mesmo número de plantas F2 com um fenótipo de tipo selvagem. </li><li> Cobrar de cada planta um disco foliar (0,60 mm) com um perfurador de furo. Odisco de folha devem ser coletadas a partir de folhas da mesma idade para ter certeza de ter quantidades similares de DNA. As amostras podem ser processadas para a extracção genómico separadamente ou em grupos. Neste caso, é possível recolher amostras 5-10 para cada tubo de Eppendorf de modo que no final terá tubos eppendorf menos a partir do qual o ADN genómico tem de ser extraído. </li><li> Planta MasterPure da folha de DNA Purificação Kit (Epicentre) é utilizado para extrair o ADN genómico. O DNA genómico obtido a partir de cada amostra é quantificada com um nanodrop. </li><li> A mesma quantidade de ADN genómico a partir de cada amostra é então colocada em conjunto, até um total de 300 ng (~ uL 30) de DNAs de plantas; adicionar 60 uL 2.5X solução iniciadores aleatórios [125 mM Tris-HCl (pH 6,8), 12,5 mM MgCl2, 25 mM 2-mercaptoetanol, 750 ug / ml iniciadores oligodeoxyribonucleotide (octâmeros aleatórios)] (Bioprime kit) e 42 uL de água (volume final de 132 uL). </li><li> DNAs desnaturar a&gt; 95 ° C durante 5 a 10 min. </li><li> Esfriar em gelo. </li><li> Para cada denatuvermelho DNA adicionar 15 uL mistura dNTPs 10X com biotina dCTP [1 mM de biotina-14-dCTP, 1 mM de dCTP, 2 mM de dATP, 2 mM de dGTP, dTTP 2 mM em 10 mM Tris-HCl (pH 7,5), 1 mM de Na2EDTA] (Bioprime kit), e completar com 3 polimerase Klenow uL (kit Bioprime). </li><li> Incubar durante a noite a 25 ° C. </li><li> No dia seguinte, adicionar 15 uL 3 M NaOAc e 400 uL frio EtOH a 100% e misturar. </li><li> Incubar a -80 ° C durante 1 hora, em seguida, girar à 20.500 xg durante 15 min, remover o sobrenadante, e lavou-se com 500 EtOH frio uL de 75%. </li><li> Girar em 20.500 xg durante 10 min. </li><li> Pelotas de matéria seca de ADN a 37 ° C durante 10 min e ressuspender em água a 100 uL. </li><li> Use 5 uL de verificar o rendimento ea qualidade sobre um gel (Figura 2). </li><li> Enviar 95 uL de etiquetagem de reacção para o Arabidopsis GeneChip de tipo selvagem e mutante para ATH1 Hibridação matriz Genoma. </li><li> As matrizes são digitalizados e os arquivos. CEL obtidos são analisados ​​utilizando o software R ( <a href="http://www.r-project.org/">http://www.r-project.o</a>rg /). </li><li> Instale o software R, em seguida, abra o programa e cole o seguinte texto: fonte (&quot; <a href="http://bioconductor.org/biocLite.R">http://bioconductor.org/biocLite.R</a> &quot;) biocLite () Em seguida, pressione retorno chave esta irá instalar os pacotes padrão (BioConductor <a href="http://www.bioconductor.org">http://www.bioconductor.org</a> ). </li><li> Em uma nova pasta no seu desktop, baixar no seguinte site ( <a href="http://www.naturalvariation.org/methods">http://www.naturalvariation.org/methods</a> ) os arquivos: readcel.R SFP.R Map.R ath1V5.RData ColLerCEL.zip O ColLerCEL.zip arquivo deve ser descompactado e os arquivos resultantes colado em sua nova pasta. </li><li> Renomeie os dados obtidos a partir de sua experiência em GeneChip wildtype.CEL e mutant.CEL. </li><li> Copie agora os seus dados de GeneChip (wildtype.CEL emutant.CEL) em sua pasta. </li><li> Abrir R, a) para Mac: clique em &quot;Outros&quot;, e clique em &quot;Altere o diretório de trabalho.&quot; b) para PC: clique em &quot;Arquivo&quot; e depois &quot;Altere o diretório&quot; </li><li> a) para Mac: Selecione a pasta que contém os arquivos. Clique em &quot;Área de Trabalho&quot;, &quot;arquivo de espaço de trabalho Load&quot; e selecione ath1V5.RData, b) para PC: Selecione a pasta que contém os arquivos acima, clique em &quot;Arquivo&quot;, depois &quot;espaço de trabalho Load&quot; e selecione ath1V5.RData. </li><li> ReadCEL.R aberto usando o Notepad e copiar o texto todo para R. </li><li> SFP.R aberto usando o Notepad e copiar o texto todo para R. </li><li> Map.R aberto usando o Notepad e copiar o texto todo para R. </li><li> Na janela do console, você verá a seguinte mensagem: X cromossomo limita Mb yz Pesquisar em genes TAIR colar neste link 3 <a href="http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y&amp;end=z">http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y</a>&amp; Fim = z </li><li> Copie e cole o link em seu navegador de internet. Uma janela irá aparecer e pedir para abrir ou salvar o arquivo, você deve selecionar salvar. Escolha um nome de arquivo e anexar a extensão &quot;. Xls&quot;, clique em Salvar. </li><li> Abra o arquivo &quot;. Xls&quot;, onde você pode encontrar a coordenada para o seu mutante (o resultado é representado graficamente na Figura 3). </li><li> Dentro da área mapeada no Illumina montado lê do genoma mutante identificar transições específicas EMS 4 entre os polimorfismos de nucleotídeos únicos. Complementar o fenótipo mutante por transformação com o gene de tipo selvagem (s) usando procedimentos padrão 15. </li></ol> 4. Os resultados representativos A Figura 1 mostra a abordagem utilizada para a identificação de um mutante do via secretora utilizando microscopia confocal de triagem. A Figura 2 mostra uma preparação típica de rotulado ADN genómico para hibridação matriz. Na figura 3, um resultado típico esperado após a análise dos dados obtidos a partir da matriz do Genoma GeneChip Arabidopsis ATH1 é apresentado. <img alt="A Figura 1" src="/files/ftp_upload/3809/3809fig1.jpg" /> Figura 1. Arabidopsis plantas transgénicas que expressam ssGFPHDEL (ER marcador) (1), foram cultivadas para produzir sementes suficientes para EMS tratamento (2). Sementes tratadas com EMS foram então semeadas para gerar plantas M1 (3). Cada planta M1 representa uma linha diferente e sementes foram recolhidos separadamente de cada um deles. Semente M2 foram plaqueadas em ½ substrato MS (4) e, em seguida, rastreados por defeitos na morfologia ER por microscopia confocal (5). Durante o rastreio encontramos plantas que conservada do tipo selvagem morfologia ER (6) e plantas que mostram defeituosos fenótipos ER (7). Estas plantas foram cultivadas para obter a geração M3 e para confirmar o fenótipo (8). DNA genómico de planta M3 foi utilizado para Solexa Illumina sequenciação (a). A mesma plantatambém foi utilizado para os cruzamentos com LER-em peso a obtenção de uma população de mapeamento F2 (b). <img alt="A Figura 2" src="/files/ftp_upload/3809/3809fig2.jpg" /> Figura 2. Bioprime reacções de rotulagem aleatórios (5 uL de 100 uL) foram carregadas em gel de agarose a 1%. Lane, um é de um pool de tipo selvagem plantas F2, e Lane b é a partir de um conjunto de mutantes plantas F2. (Marcador é 1 Kb de DNA escada-N3232, a partir de NEB). <img alt="A Figura 3" src="/files/ftp_upload/3809/3809fig3.jpg" /> Figura 3. A figura representa um exemplo de mapeamento de Col-0 mutação usando GeneChip Arabidopsis ATH1 Hibridação Matriz Genoma. A mutação neste exemplo está localizado no cromossoma 1 delimitado, por barras verticais. As barras horizontais representam os limiares para detecção.

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Aqui, foi descrito um rastreio de microscopia confocal baseado na identificação de mutantes endomembrane. Esta abordagem pode ser facilmente adaptado para outras organelas da célula para os quais marcadores específicos de proteínas fluorescentes estão disponíveis. A tela é baseado na identificação de mutantes que mostram uma distribuição aberrante do marcador fluorescente, quer na organela alvo ou para organelas que não são supostos para conter o marcador. Respectivamente, estes mutantes representam popula...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Sulfonato Ethylmethane </td><td> Sigma </td><td> M0880 </td></tr><tr><td> NaOH </td><td> JT Baker </td><td> 3722-05 </td></tr><tr><td> Murashige Skoog basais médios w Gamborg vitaminas </td><td> Phyto Technolog laboratorie </td><td> M404 </td></tr><tr><td> Phytagel </td><td> Sigma </td><td> P8169-1 kg </td></tr><tr><td> Mini kit RNeasy </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> Mestre pura planta folha kit de purificação de DNA </td><td> Epicentro </td><td> MPP92100 </td></tr><tr><td> Sistema de rotulagem Bioprime DNA </td><td> Invitrogen </td><td> 18094-011 </td></tr><tr><td> Prova do álcool 200 </td><td> Decan Laboratories Inc. </td><td> 2716 </td></tr><tr><td> NaOAc </td><td> JT Baker </td><td></td></tr><tr><td> Gene ATH1 Arabidopsis chipsmatriz genoma </td><td> Affymetrix </td><td> 900385 </td></tr><tr><td> Falcon tubos 50 mL </td><td> corning </td><td> 430290 </td></tr><tr><td> Tubos Eppendorf de 1,5 mL </td><td></td><td></td></tr><tr><td> Papel de filtro 90 milímetros </td><td> Whatman </td><td> 1001090 </td></tr><tr><td> Balança Analítica </td><td> Mettler Toledo AB54-S </td><td> nd </td></tr><tr><td> Nutação (onda) shaker </td><td> Heidolph PolyMax 1040 </td><td> nd </td></tr><tr><td> Centrifugar </td><td> Eppendorf 5417-R </td><td> nd </td></tr></tbody></table>

fluorescence-microscopy screening and next-generation sequencing: useful tools for the identification of genes involved in organelle integrity

Este protocolo descreve um rastreio microscópio de fluorescência baseada de plântulas de Arabidopsis e descreve como mapear mutações recessivas que alteram a distribuição subcelular de um marcador específico marcado fluorescente na via secretora. Arabidopsis é um poderoso modelo biológico para estudos genéticos devido ao seu tamanho de genoma, tempo de geração e conservação de mecanismos moleculares entre os reinos. A matriz de genotipagem como uma abordagem para mapear a mutação em alternativa ao método tradicional baseado em marcadores moleculares é vantajosa porque é relativamente rápido e pode permitir que o mapeamento de vários mutantes de um período de tempo muito curto. Este método permite a identificação de proteínas que podem influenciar a integridade de qualquer organela em plantas. Aqui, como um exemplo, é proposto um ecrã para mapear genes importantes para a integridade do retículo endoplasmático (RE). Nossa abordagem, no entanto, pode ser facilmente estendido para outras organelas celulares vegetais(Por exemplo, ver 1,2), e, portanto, representa um passo importante para compreender a base molecular que regula outras estruturas subcelulares.

Watch this Scientific Journal Video about Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity at JoVE.com

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

Uma busca fundamentais em biologia celular é definir os mecanismos subjacentes à identidade dos organelos que tornam as células eucarióticas. Aqui é proposto um método para identificar os genes responsáveis ​​pela integridade morfológica e funcional de organelas vegetais utilizando microscopia de fluorescência e as ferramentas a próxima geração de sequenciação.

Microscopia de fluorescência de Triagem e seqüenciamento da próxima geração: Ferramentas úteis para a identificação de genes envolvidos na integridade das Organelas

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

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12.2K visualizações.  Michigan State University. Uma busca fundamentais em biologia celular é definir os mecanismos subjacentes à identidade dos organelos que tornam as células eucarióticas. Aqui é proposto um método para identificar os genes responsáveis ​​pela integridade morfológica e funcional de organelas vegetais utilizando microscopia de fluorescência e as ferramentas a próxima geração de sequenciação.

Vídeo: Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Lapso de tempo de imagens de Mitose Depois de Transfection siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Biologia

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Usando RNA mediada estratégia de alimentação Interferência em tela para genes envolvidos na regulação do tamanho corporal na Nemátodo<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

O sequenciamento de próxima geração de 16S RNA ribossomal Gene amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

Alvejado DNA análise de metilação por Sequenciação de próxima geração

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Rastreio de alto rendimento para moduladores químicos de Genes Post-transcriptionally Regulamentados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplificação, sequenciamento de próxima geração, e Genomic DNA Mapeamento das retrovirais Integração Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Interactome-Seq: Um protocolo para a construção da biblioteca de Domainome, a validação e a seleção por Phage Display e a próxima geração de sequenciamento

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

Caracterização de Microbiome carrapato de próxima geração 16S rRNA Amplicon sequenciação

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

Análise de microbiota usando PCR de duas etapas e sequenciamento do gene 16S rRNA de próxima geração

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

Um protocolo de extração de DNA de mosquito baseado em contas magnéticas para sequenciamento de próxima geração

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...