O trabalho é apoiado por uma concessão do Instituto de Desenvolvimento do Hospital Infantil da Filadélfia.

1. Cruz-link Chromatin <ol><li> Cultivar células em placas de cultura de 100x20mm celulares. A quantidade de células pode variar de 1 a 10 milhões de células por placa, dependendo do tipo de célula. Aproximadamente 2 milhões de células é suficiente para uma imunoprecipitação. </li><li> Células de ligação cruzada em formaldeído a 1% durante 10 min à temperatura ambiente com agitação ocasional. </li><li> Quench de reticulação por adição de uma concentração final de glicina 125 mM e incubar durante 5 min à temperatura ambiente. </li><li> Lavar as células com solução salina tamponada de fosfato 1X (PBS) duas vezes, decantar PBS e, em seguida, adicionar 0,2 ml de PBS. </li><li> Células de colheita com um raspador de células de plástico dentro de um tubo de microcentrífuga. </li><li> Girar as células a 2.000 rpm durante 5 min a 4 ° C. </li><li> Aspirar o sobrenadante. Ressuspender as células em tampão de lise (SDS a 1% de SDS, 10 mM de EDTA, 50 mM Tris-HCl pH 8,1) para a célula lisado ou mantê-los como uma pastilha para a extracção nuclear. </li><li> As células podem ser guardadas a -80 ° C, ou pode-se proceder immédiately com sonicação. </li></ol> 2. Prepare Núcleos (Avance para o Passo 3.5 para lisado celular inteiro) <ol><li> Suplemento de Lise Celular (5 mM de PIPES, pH 8,0, KCl 85 mM, 0,5% de NP-40) com 1X Inibidor de Protease de cada experimento. </li><li> Ressuspender pellet celular descongelado em cerca de 10 vezes o volume de pelotas com Tampão de Lise Celular. </li><li> DOUNCE-homogeneizar 10 vezes com pilão, em seguida, incubar no gelo por 10 min. </li><li> Centrifugar amostra a 4000 rpm durante 5 min a 4 ° C, rejeitar o sobrenadante e guardar pelete nuclear. </li></ol> 3. Sonication * <ol><li> Aqueça SDS Tampão de Lise e quantidade de suplemento de tampão a ser utilizado com inibidor de proteinase. </li><li> Ressuspender pellet nuclear em SDS Lysis Buffer (aproximadamente 0,5 ml de tampão por 1-10.000.000 células) </li><li> Incubar no gelo por 10 min. </li><li> Adicionar 0,5 mL de alíquotas de amostras para tubos de microcentrífuga. </li><li> Sonicar em gelo húmido utilizando ultra-sons Misonix com 30 segundos ligado e 45 segundosfora com uma definição de amplitude de 2. O número de ciclos de tamanho ideal fragmento pode ser determinada pela primeira experimentar vários números de ciclo (ex. 2, 4, 8, 12, 16, e 20 ou mais ciclos). Um tipo diferente de ultrassons pode ser usada, no entanto, as condições podem variar. Experimentação com o número de ciclos e da quantidade de tempo ligado e desligado devem ser realizadas a fim de determinar as condições ideais. </li><li> Coletar 20 ml de cada amostra para verificar os resultados sonicação e fazer a quantificação,. O restante da amostra pode ser armazenada a -80 ° C. </li><li> Dilui-se a 20 uL da amostra por adição de 30 ul de 0,1 x tampão TE. </li><li> Tratar amostra com 1 ml de RNase A a 37 ° C durante 1 hora, em seguida adicionar 1 ul de proteinase K, e incubar a 62 ° C durante 2 horas. </li><li> Executar 20 ul da amostra num gel de agarose a 2%. </li><li> Purificar o restante da amostra com QIAquick kit de purificação de PCR, em seguida, quantificar usando espectrofotômetro NanoDrop. </li></ol> * Para ChI nativaP, microccocal nuclease de digestão pode ser usado como alternativa ao corte do DNA. 4. Bloco de agarose * <ol><li> Se contas já estão bloqueadas, vá para o passo 5.1. </li><li> É uma proteína ou Proteína G agarose. Por 5 imunoprecipitações (IPs), use 600 ml de 50% chorume talão (300 ml talão pellet) </li><li> Para lavar as contas, colocá-los para baixo a 800 rpm por 1 min a 4 ° C e descartar o sobrenadante. Adicionar um pouco mais do que 2 ml de Tampão de Diluição do chip (0,01% de SDS, EDTA 1,2 mM, NaCl 167 mM, 1,1% de Triton X-100, 16,7 mM Tris-HCl pH 8,1) e misturar lentamente invertendo o tubo de 10X. Spin-se novamente a 800 rpm durante 1 min a 4 ° C e desprezar o sobrenadante. Repetir a lavagem duas vezes mais. </li><li> Bloquear a grânulos rodando a 4 ° C durante a noite em solução de bloqueio. Consulte a Tabela 1 para a receita da solução de bloqueio. </li></ol> 5. Cromatina pré-clear <ol><li> Thaw sonicado cromatina no gelo. </li><li> Girar a 12.000 rpm fou 10 min a 4 ° C, em seguida, colocar no gelo imediatamente para remover o SDS (bolinha branca). </li><li> Recolher o sobrenadante, descarte da pelota, e combinar as amostras, se necessário. </li><li> Retire os montantes necessários para o experimento com base em cálculos (1-10 ug da cromatina por IP). </li><li> Diluir cromatina 10X em Chip tampão de diluição suplementada com inibidor de protease. </li><li> Adicionam-se 100 ul de contas bloqueadas por IP. </li><li> Girar a 4 ° C durante 1 hora. </li></ol> 6. Immunoprecipitation <ol><li> Girar as amostras a 800 rpm durante 1 min, e transferir o sobrenadante para um tubo fresco. </li><li> Girar o sobrenadante a 800 rpm durante 1 min e transferência para outro tubo limpo. </li><li> Guardar 20 ul de sobrenadante, para servir como controlo de entrada, a -20 ° C. </li><li> Alíquota da cromatina com o número de IPs a serem feitas no experimento. </li><li> Adicionam-se 2 ug de anticorpo por 1-10 ug de cromatina para cada amostra. </li><li> Incubar durante a noite de 4 ° C com rotação. </li><li> Adicionam-se 100 ul de contas bloqueadas para cada amostra de IP. </li><li> Incubar durante 1 hora a 4 ° C com rotação. </li><li> Agregar as esferas para baixo, girando a 800 rpm durante 1 min e descartar tanto do sobrenadante quanto possível. </li><li> Lavar contas uma vez com Low tampão de lavagem complexo imunológico sal. Adicionar 1 ml de tampão para cada tubo, girar à temperatura ambiente durante 5-8 minutos; girar para baixo a 800 rpm durante 1 min, em seguida, descartar o sobrenadante. Repita a lavagem uma vez com tampão de sal elevado complexo imune de lavagem e o tampão de lavagem complexo imunológico LiCl e duas vezes com tampão TE para um total de cinco lavagens (Tabela 2). </li></ol> 7. Eluição <ol><li> Descongelar amostras de entrada a partir do dia anterior a ser processadas com os eluentes. </li><li> Faça eluição tampão fresco (Tabela 3). </li><li> Faça uma mistura de mestre suficiente tampão de eluição necessário para IPs e amostras de controle de entrada mais 1-2 amostras extras. </li><li> Adicionar 100 ul de tampão de eluição para cada uma das amostras de IP e incubar à temperatura ambiente durante 15 mcom rotação. </li><li> Girar a 800 rpm durante 1 min e adicionar o sobrenadante para um tubo fresco. </li><li> Adicionar mais 100 ul de tampão de eluição a cada tubo de esferas e incubar à temperatura ambiente durante 15 minutos com rotação. </li><li> Vortex durante 15 segundos após a incubação; girar a 5000 rpm, durante 1 min, depois combinar o sobrenadante com o sobrenadante da primeira eluição. (Certifique-se que não há sobra contas no sobrenadante. Caso de dúvida, girar sobrenadante novamente a 5.000 rpm por 1 min e recolher o sobrenadante em um novo tubo. </li><li> Adicionar 180 ul de tampão de eluição à 20 ul das amostras de controlo de entrada. </li></ol> 8. Cross-link reverso <ol><li> Para a 200 ul de eluentes e controles de entrada, adicionar 8 mL de 5 M de NaCl. </li><li> Tubos de vedação com parafilme e incubar em banho-maria a 65 ° C durante a noite. </li></ol> 9. Purificação de DNA <ol><li> Tratar cada amostra com 1 ml de RNase A durante 1 hora a 37 ° C. </li><li> Adicionar 4 μl de EDTA 0,5 M, 8 ul de 1M Tris-HCl, misturar, adicionar 1 ml de Proteinase K a cada amostra e incubar a 45 ° C durante 2 horas. </li><li> Purifica-se as amostras usando o kit de purificação de PCR QIAquick. As amostras podem ser guardadas a -20 ° C e a PCR de verificação pode ser feito numa data posterior. </li></ol> * Alternativamente, as esferas magnéticas ChIP grau pode ser usado em lugar de agarose para a porção de imunoprecipitação. 10. PCR cheque <ol><li> Para a verificação de PCR, use primers para regiões conhecidas em se comprometer com a proteína de interesse. Além disso, use primers para regiões não-vinculativos como controles negativos. </li><li> Misturar os reagentes para a reacção. Diluir a amostra de entrada a 1:100 (Tabela 3). </li><li> Executar reação. Programa de PCR: </li></ol> Passo 1: 94 ° C 3 min Passo 2: 94 ° C 20 seg 59 ° C 30 seg 72 ° C 30 seg (Repita a Etapa 2, pelo menos, 30 cycles) Passo 3: 72 ° C 2 min <ol start="4"><li> Executar amostras em gel de agarose a 1%. </li><li> O enriquecimento pode também ser determinada quantitativamente, por PCR em tempo real. </li></ol>

<ol>
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Os autores declaram que não têm interesses financeiros concorrentes.

É agora possível realizar um perfil de todo o genoma de ADN-proteína utilizando as interacções associação ChIP seq, como foi recentemente demonstrado com outros factores de transcrição de 2,3. A chave para um resultado bem sucedido de sequenciação é a geração de um modelo de alta qualidade de ADN de cromatina imunoprecipitação. Uma vez que o molde de ADN foi gerado e verificado ser adequadamente enriquecida, pode-se, em seguida, levá-lo na preparação de bibliote...

Durante muitos anos, tem havido uma necessidade não satisfeita para identificar o grupo de genes ligados e regulada por um genoma proteína dada largura, em particular os da classe factor de transcrição. Odom et al. 1 usado cromatina imunoprecipitação (CHIP), combinado com o promotor de microarrays para identificar sistematicamente os genes ocupados por órgãos reguladores de transcrição pré-especificados em ilhotas pancreáticas fígado humano e. Subsequentemente, Johnson et ai. Dois desenvolveram um ensaio de imunoprecipitação da cromatina em larga escala com base em ultra-sequenciação de DNA de alto rendimento directo (ChIP seguintes), a fim de mapear de maneira abrangente interacções proteína-ADN em toda genomas de mamíferos. Tal como um caso de teste, eles mapearam in vivo a ligação do factor de silenciador neurónio restritiva (NRSF) para 1946 locais no genoma humano. Os dados exibidos sharp resolução da posição de ligação (+ 50 pares de bases), o que facilitou tanto o isolation de motivos ea identificação dos motivos NRSF vinculativas. Estes dados do chip-seq também teve alta sensibilidade e especificidade e confiança estatística (P &lt;10 -4), propriedades que são importantes para inferir novas interações candidatos. Robertson et al. 3 também usado ChIP seq para mapear STAT1 alvos no interferão-γ (IFN-γ)-estimuladas e não estimuladas As células HeLa S3 humanas in vivo. Por Chip-seq, com 15,1 e 12,9 milhões seqüência mapeada unicamente lê, e uma taxa de falsa descoberta estimada em menos de 0.001, eles identificaram 41.582 e 11.004 STAT1 regiões de ligação putativos em células estimuladas e não estimuladas, respectivamente. Dos 34 loci conhecidos para conter STAT1 interferon-responsive ligação 4-8 locais, Chip-seq encontrados 24 (71%). Alvos ChIP-seq foram enriquecidos em sequências semelhantes aos conhecidos STAT1 motivos de ligação. Comparações com dois dados do chip-PCR existentes define sugeridoque a sensibilidade ChIP seq situou-se entre 70% e 92% e especificidade de, pelo menos, 95%. Além disso, ficou claro que o chip-seq oferece baixa complexidade analítica e sensibilidade que aumenta com a profundidade de seqüenciamento. Como as tecnologias de seqüenciamento tal, genoma &quot;next-generation&quot; fornecer 1-2 ordens de magnitude aumento na quantidade de seqüência que pode ser custo-benefício gerado sobre as tecnologias mais velhos 9. Métodos ChIP-seq, portanto, fornecer diretamente a cobertura de todo o genoma para profiling eficaz de mamíferos interações DNA-proteína 3. Em 2006, uma forte associação de variantes do fator de transcrição 7-like 2 (TCF7L2) gene com diabetes tipo 2 foi descoberto 10. Outros pesquisadores já replicado de forma independente este achado em diferentes etnias e, curiosamente, desde os primeiros estudos de associação ampla do genoma de diabetes tipo 2 publicados na revista Nature 11,12 </saté&gt;, Ciência 13-15 e em outros lugares 16,17, a associação mais forte foi de fato com TCF7L2, o que é agora considerado o mais importante descoberta genética na diabetes tipo 2 até à data 18-20. Além disso, TCF7L2 tem sido associada a risco de cancro 21,22; facto, esta ligação tornou-se mais evidente quando o locus 8q24 revelado pelos estudos genoma amplo associação de um certo número de cancros, incluindo os carcinomas colorrectais, foi demonstrado ser devido a um extremo a montante TCF7L2 elemento de ligação dirigir a transcrição de MYC 23,24. Como tal, há um grande interesse em determinar os genes a jusante regulados por este factor de transcrição chave. Com base na experiência com TCF7L2 como um exemplo do método, este documento descreve como gerar o molde de ADN de chip de alta qualidade. Chip foi realizada na linha celular de carcinoma colo-rectal, HCT116, para sequenciação subsequente, a fim construir uma alta resolmapa buição dos genes vinculados por TCF7L2 25 em um esforço para produzir mais conhecimento para o seu papel fundamental na patogênese de características complexas.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td></td>
 </tr>
 <tr>
 <td>QIAquick PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28104 </td>
 <td></td>
 </tr>
 <tr>
 <td>EZ-ChIP Kit </td>
 <td>Millipore </td>
 <td>17-371 </td>
 <td></td>
 </tr>
 <tr>
 <td>GoTaq Hot Start Polymerase </td>
 <td>Promega</td>
 <td>M5001</td>
 <td></td>
 </tr>
 <tr>
 <td>Misonix Sonicator</td>
 <td>Qsonica </td>
 <td>XL-2000 </td>
 <td></td>
 </tr>
 <tr>
 <td>
 NanoDrop 1000 Spectrophotometer </td>
 <td>
 Thermo-Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Positive control primer sequences (TCF7L2-1) 
 Forward- 5'-TCGCCCTGTCAATAATCTCC-3' 
 Reverse- 5'-GCTCACCTCCTGTATCTTCG-3' 
 Negative control primer sequences (CTRL-1) 
 Forward-5'-ATGTGGTGTGGCTGTGATGGGAAC-3'
 
 Reverse- 5'-CGAGCAATCGGTAAATAGGTCTGG-3'</td>
 </tr>
 </tbody>
</table>

generation of high quality chromatin immunoprecipitation dna template for high-throughput sequencing (chip-seq)

Microplaqueta-seqüenciamento (Chip-seq) métodos diretamente oferecer cobertura de todo o genoma, onde a combinação de cromatina imunoprecipitação (CHIP) e sequenciamento massivamente paralelo pode ser utilizado para identificar o repertório de sequências de DNA de mamíferos vinculados por fatores de transcrição in vivo. &quot;Próxima geração&quot; tecnologias de seqüenciamento do genoma fornecer 1-2 ordens de magnitude aumento na quantidade de seqüência que pode ser rentável gerou mais de tecnologias mais antigas permitindo métodos ChIP-seq para fornecer diretamente a cobertura de todo o genoma para profiling eficaz de mamíferos interacções proteína-ADN. Para o sucesso abordagens ChIP-seq, deve-se gerar modelo de DNA chip de alta qualidade para obter os melhores resultados de sequenciamento. A descrição é baseada em experiência com o produto de proteína do gene mais fortemente implicado na patogénese da diabetes de tipo 2, a saber, o factor de transcrição do factor de transcrição 7-like 2 (TCF7L2). Este factor também tem sido implicado em vários cancros. Descritos é como ADN molde para gerar chip de alta qualidade derivada da linha celular de carcinoma colo-rectal, HCT116, a fim de construir um mapa de alta resolução através de sequenciação para determinar os genes ligados por TCF7L2, dando uma visão mais para o seu papel chave na patogénese de características complexas.

Uma vez que a cromatina foi sonicada e foram tratados com RNase e Proteinase, as amostras são executados em gel de agarose a 2%, devem apresentar um esfregaço com a maior parte do ADN com o tamanho desejado. Se vários ciclos diferentes são testadas, uma redução gradual no tamanho deve ser visto como aumentar o número de ciclos (Figura 2). Depois de completar a porção de imunoprecipitação do protocolo do enriquecimento pode ser verificada por PCR, ou PCR em tempo r...

Watch this Scientific Journal Video about Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq at JoVE.com

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

A combinação de cromatina imunoprecipitação e sequenciamento ultra-high-throughput (CHIP-seq) pode identificar e mapear as interações DNA-proteína em uma determinada linha de tecido ou célula. Delineado é como gerar um modelo de alta chip de qualidade para o seqüenciamento posterior, utilizando a experiência com o fator de transcrição TCF7L2 como um exemplo.

Geração de alta Chromatin Template DNA Immunoprecipitation Qualidade para seqüenciamento de alto rendimento (CHIP-seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

generation-high-quality-chromatin-immunoprecipitation-dna-template

Research

JoVE Journal

Biology

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

24.3K visualizações.  Children's Hospital of Philadelphia Research Institute. A combinação de cromatina imunoprecipitação e sequenciamento ultra-high-throughput (CHIP-seq) pode identificar e mapear as interações DNA-proteína em uma determinada linha de tecido ou célula. Delineado é como gerar um modelo de alta chip de qualidade para o seqüenciamento posterior, utilizando a experiência com o fator de transcrição TCF7L2 como um exemplo.

Vídeo: Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

Ligadura altamente eficiente de pequeno RNA moléculas microRNA para quantificação por sequenciamento de alto rendimento

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Biologia

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 ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Ensaio para imunoprecipitação da cromatina a identificação de Arabidopsis interações proteína-ADN<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-seq: Genoma-wide Mapeamento das Interações Proteína-DNA por High Throughput Sequencing de fragmentos de DNA metilado-Adenina

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

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 ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

De alto rendimento CRISPR Vector construção e caracterização de DNA Modificações pela geração de raízes de tomateiro Cabeludo

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

Um Protocolo de Avaliação Funcional da Whole-Protein saturação mutagênese Bibliotecas Utilizando-High Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Ensaio cinético de molécula única de alta produtividade paralela para decote de DNA específico do local

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

Métodos modificados para carregamento de placas de extração de DNA de alto rendimento reduzem o potencial de contaminação

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...