El trabajo es apoyado por una concesión del Instituto de Desarrollo del Hospital de Niños de Filadelfia.

1. Cross-link cromatina <ol><li> Se cultivan las células en placas de cultivo celular 100x20mm. La cantidad de células puede variar de 1 a 10 millones de células por plato dependiendo del tipo de célula. Aproximadamente 2 millones de células es suficiente para una inmunoprecipitación. </li><li> Células Cross-link en el 1% de formaldehído durante 10 minutos a temperatura ambiente con agitación ocasional. </li><li> Se inactiva la reticulación mediante la adición de una concentración final de 125 mM de glicina y se incuba durante 5 min a temperatura ambiente. </li><li> Lavar las células con solución salina tamponada con fosfato 1X (PBS) dos veces, se decanta PBS, y a continuación, añadir 0,2 ml de PBS. </li><li> Células de la cosecha con un rascador de células de plástico en un tubo de microcentrífuga. </li><li> Centrifugar las células a 2000 rpm durante 5 min a 4 ° C. </li><li> Aspirar el sobrenadante. Resuspender las células en tampón de lisis (SDS al 1% de SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8,1) para lisado de células enteras o mantenerlos como una pastilla para la extracción nuclear. </li><li> Las células se pueden guardar a -80 ° C o se puede proceder immediately con ultrasonidos. </li></ol> 2. Prepare Núcleos (Continúe con el paso 3.5 para Todo el lisado celular) <ol><li> Suplemento Tampón de Lisis Celular (TUBOS 5 mM, pH 8,0, 85 mM de KCl, 0,5% de NP-40) con 1X inhibidor de la proteinasa cada experimento. </li><li> Resuspender el sedimento celular descongelado en aproximadamente 10 veces el volumen de pellets con Tampón de Lisis Celular. </li><li> Dounce-homogeneizar 10 veces con mano de mortero y luego se incuban en hielo durante 10 min. </li><li> Centrifugar la muestra a 4000 rpm durante 5 min a 4 ° C, desechar el sobrenadante, y guardar pellet nuclear. </li></ol> 3. Sonicación * <ol><li> Calentar SDS tampón de lisis y la cantidad de suplemento de tampón para ser utilizado con el inhibidor de proteinasa. </li><li> Resuspender el sedimento nuclear en SDS tampón de lisis (aproximadamente 0,5 ml de tampón por 1 a 10.000.000 células) </li><li> Incubar en hielo durante 10 min. </li><li> Añadir alícuotas de 0,5 ml de las muestras a tubos de microcentrífuga. </li><li> Someter a ultrasonidos en hielo usando Misonix ultrasonidos con 30 segundos encendido y 45 segundosbajar en un ajuste de amplitud de 2. Número de ciclos para el tamaño ideal fragmento puede ser determinada por tratando primero a cabo varios números de ciclos (por ejemplo 2, 4, 8, 12, 16, y 20 o más ciclos). Una marca diferente de aparato de ultrasonidos puede utilizarse, sin embargo, las condiciones pueden variar. La experimentación con el número de ciclos y la cantidad de tiempo de encendido y apagado deben llevarse a cabo con el fin de determinar las condiciones ideales. </li><li> Recoge 20 l de cada muestra para comprobar los resultados de sonicación y hacer cuantificación. El resto de la muestra puede ser almacenada a -80 ° C. </li><li> Se diluye la 20 l de muestra mediante la adición de 30 l de 0,1 X tampón TE. </li><li> Tratar la muestra con 1 l de RNasa A a 37 ° C durante 1 hr a continuación, añadir 1 l de proteinasa K y se incuba a 62 ° C durante 2 horas. </li><li> Ejecutar 20 l de la muestra en un gel de agarosa al 2%. </li><li> Purificar la cantidad restante de la muestra con el kit de purificación de PCR QIAquick entonces cuantificar utilizando espectrofotómetro NanoDrop. </li></ol> * Para ChI nativaP, microccocal la digestión con nucleasa puede usarse alternativamente para cortar el ADN. 4. Cuentas bloque de agarosa * <ol><li> Si los granos ya están bloqueados, vaya al paso 5.1. </li><li> Utilice una proteína o proteína G agarosa. Para 5 inmunoprecipitaciones (IPs), utilice 600 l de 50% de suspensión de perlas (300 l sedimento de perlas) </li><li> Para lavar las perlas, girar hacia abajo a 800 rpm durante 1 min a 4 ° C y desechar el sobrenadante. Añadir un poco más de 2 ml de tampón de dilución de chip (0,01% de SDS, 1,2 mM de EDTA, 167 mM de NaCl, 1,1% de Triton X-100, Tris-HCl 16,7 mM, pH 8,1) y mezclar lentamente invirtiendo el tubo 10 veces. Girar de nuevo a 800 rpm durante 1 min a 4 ° C y desechar el sobrenadante. Repita el lavado 2 veces más. </li><li> Bloque perlas por rotación a 4 º C durante la noche en solución de bloqueo. Consulte la Tabla 1 para la receta de la solución de bloqueo. </li></ol> 5. Cromatina Pre-clear <ol><li> Thaw ultrasonidos cromatina en hielo. </li><li> Centrifugar a 12.000 rpm fo 10 min a 4 ° C y luego se puso en hielo inmediatamente para eliminar el SDS (blanco pellet). </li><li> Recoger el sobrenadante, deseche pellet y combinar las muestras si es necesario. </li><li> Sacar las cantidades necesarias para el experimento sobre la base de cálculos (1-10 ug de la cromatina por IP). </li><li> Diluir 10 veces la cromatina en el tampón de dilución chip complementado con inhibidor de la proteinasa. </li><li> Añadir 100 l de cuentas bloqueadas por IP. </li><li> Girar a 4 ° C durante 1 hora. </li></ol> 6. Immunoprecipitation <ol><li> Centrifugar las muestras a 800 rpm durante 1 min y transferir el sobrenadante a un tubo nuevo. </li><li> Decantar el sobrenadante a 800 rpm durante 1 min y la transferencia a otro tubo limpio. </li><li> Guardar 20 l del sobrenadante, para servir como el control de entrada, a -20 ° C. </li><li> Alícuota de la cromatina en el número de direcciones IP que se harán en el experimento. </li><li> Añadir 2 g de anticuerpo por ug 1-10 de la cromatina a cada muestra. </li><li> Incubar toda la noche 4 º C con rotación. </li><li> Añadir 100 l de cuentas bloqueadas a cada muestra IP. </li><li> Incubar durante 1 hora a 4 ° C con rotación. </li><li> Pellet las perlas por hilatura hacia abajo a 800 rpm durante 1 min y desechar tanto del sobrenadante como sea posible. </li><li> Lávese las cuentas una vez con sal baja Inmune tampón de lavado Complex. Añadir 1 ml de tampón a cada tubo; girar a temperatura ambiente durante 5-8 min; girar hacia abajo a 800 rpm durante 1 min, a continuación, eliminar el sobrenadante. Repetir el lavado una vez con sal de alta Inmune tampón de lavado y el tampón de lavado Complejo complejo inmunológico LiCl y dos veces con tampón TE para un total de 5 lavados (Tabla 2). </li></ol> 7. Elución <ol><li> Descongelar las muestras de entrada a partir del día anterior a procesar con los eluyentes. </li><li> Hacer elución Tampón A nuevo (Tabla 3). </li><li> Hacer una mezcla maestra de suficiente tampón de elución necesario para IPs y muestras de control de entrada más 1-2 muestras adicionales. </li><li> Añadir 100 l de tampón de elución a cada muestra de IP e incubar a temperatura ambiente durante 15 mcon rotación. </li><li> Centrifugar a 800 rpm durante 1 min y añadir sobrenadante a un tubo nuevo. </li><li> Añadir otra 100 l de tampón de elución a cada tubo de bolas y se incuba a temperatura ambiente durante 15 minutos con rotación. </li><li> Vortex durante 15 segundos después de la incubación; centrifugar a 5.000 rpm durante 1 min, a continuación, combinar el sobrenadante con el sobrenadante de la primera elución. (Asegúrese de que no existen sobrantes cuentas en los sobrenadantes. Si no está seguro, la desaceleración sobrenadante de nuevo a 5.000 rpm durante 1 min y recoger el sobrenadante en un tubo nuevo. </li><li> Añadir 180 l de tampón de elución al 20 l de muestras de control de entrada. </li></ol> 8. Cross-link inversa <ol><li> Para los 200 l de eluyentes y controles de entrada, agregue 8 l de 5 M NaCl. </li><li> Tubos sello con parafilm y se incuba en un baño de agua a 65 ° C durante la noche. </li></ol> 9. Purificación de ADN <ol><li> Tratar a cada muestra con 1 l de RNasa A durante 1 hora a 37 ° C. </li><li> Añadir 4 μl de EDTA 0,5 M, 8 l 1 M Tris-HCl, la mezcla, a continuación, añadir 1 l de proteinasa K a cada muestra y se incuba a 45 ° C durante 2 horas. </li><li> Purificar las muestras usando kit de purificación QIAquick PCR. Las muestras pueden guardarse a -20 ° C y verificación PCR se puede hacer en una fecha posterior. </li></ol> * Alternativamente, perlas magnéticas chip-grado se pueden utilizar en lugar de agarosa para la porción de inmunoprecipitación. 10. Cheque PCR <ol><li> Para la comprobación de PCR, use cebadores para las regiones que se sabe están obligados por la proteína de interés. Asimismo, el uso cebadores para las regiones no vinculantes como controles negativos. </li><li> Mezcle los reactivos para la reacción. Diluir la muestra de entrada a 1:100 (Tabla 3). </li><li> Ejecutar reacción. Programa de PCR: </li></ol> Paso 1: 94 ° C 3 min Paso 2: 94 ° C 20 seg 59 ° C 30 seg 72 ° C 30 seg (Repita el paso 2 durante al menos 30 cycles) Paso 3: 72 ° C 2 min <ol start="4"><li> Corra las muestras en gel de agarosa 1%. </li><li> Enriquecimiento también se puede determinar cuantitativamente con PCR en tiempo real. </li></ol>

<ol>
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Los autores declaran que no tienen intereses financieros en competencia.

Ahora es posible llevar a cabo un genoma en todo el perfil de asociación interacciones proteína-ADN utilizando chip-ss, como se ha demostrado muy recientemente con otros factores de transcripción 2,3. La clave para un resultado exitoso secuenciación es la generación de una cromatina immunoprecipitation molde de ADN de alta calidad. Una vez que la plantilla de ADN se ha generado y se cercioró de ser enriquecido adecuada, uno puede entonces tomar en preparación biblioteca par...

Durante muchos años ha habido una necesidad no satisfecha para identificar el conjunto de genes vinculados y regulado por una proteína dada genoma de ancho, en particular los de la clase de factor de transcripción. Odom et. Al 1 utiliza la cromatina immunoprecipitation (CHIP) en combinación con el promotor microarrays para identificar sistemáticamente los genes ocupados por reguladores transcripcionales pre-especificados en el hígado humano y los islotes pancreáticos. Posteriormente, Johnson et al. 2 desarrollaron un ensayo de inmunoprecipitación de cromatina a gran escala basada en la secuenciación directa de ultra alto rendimiento de ADN (chip-ss) para asignar globalmente las interacciones proteína-ADN a través de todo los genomas de mamíferos. Como caso de prueba, se asignan in vivo la unión del factor silenciador restrictivo neuronal (NRSF) a 1.946 localizaciones en el genoma humano. Los datos muestran una nítida resolución de la posición de encuadernado (+ 50 pares de bases), lo que facilitó la isolation de motivos y la identificación de motivos NRSF vinculantes. Estos datos chip-seq también tenían una alta sensibilidad y especificidad y la confianza estadística (P &lt;10 -4), propiedades que son importantes para inferir nuevas interacciones candidatos. Robertson et al. 3 también se utiliza chip-ss con el fin de asignar STAT1 objetivos en interferón-γ (IFN-γ)-estimulado y células HeLa S3 humanos no estimulados in vivo. Por chip-ss, con 15,1 y 12,9 millones de secuencia asignado únicamente lee, y se estima una tasa de falso descubrimiento de menos de 0.001, se identificaron 41.582 y 11.004 supuestos regiones STAT1 vinculante en células estimuladas y no estimuladas, respectivamente. De los 34 loci se sabe que contienen STAT1 que responden al interferón vinculante sitios 4-8, chip-ss encontraron 24 (71%). Objetivos chip-ss fueron enriquecidos en secuencias similares a conocidos STAT1 vinculante motivos. Las comparaciones con los datos de dos chip-PCR existentes establece sugirióque la sensibilidad chip-ss fue entre 70% y 92% y la especificidad fue de al menos 95%. Además, estaba claro que el chip-ss ofrece tanto de baja complejidad analítica y la sensibilidad que aumenta con la secuenciación de profundidad. Como las tecnologías de secuenciación tal, genoma &quot;de nueva generación&quot; ofrecen 1-2 órdenes de magnitud aumento en la cantidad de secuencia que puede ser rentable generado más viejas tecnologías 9. Por lo tanto, los métodos de chip-ss proporcionan directamente la cobertura de todo el genoma de perfiles eficaz de mamíferos interacciones proteína-ADN 3. En 2006, una fuerte asociación de variantes en el factor de transcripción 7-like 2 (TCF7L2) gen con la diabetes tipo 2 fue descubierto 10. Otros investigadores ya han replicado este hallazgo independiente en diferentes etnias y, curiosamente, a partir de los primeros estudios de asociación de todo el genoma de la diabetes tipo 2, publicados en la revista Nature 11,12 </sa&gt;, Ciencia 13-15 y en otros 16,17, la asociación más fuerte fue hecho con TCF7L2, lo que ahora se considera el hallazgo genético más importante en la diabetes tipo 2 al día 18-20. Además, TCF7L2 se ha relacionado con el riesgo de cáncer 21,22, de hecho, esta conexión se hizo más evidente cuando el locus 8q24 revelado por estudios de asociación en todo el genoma de un número de cánceres, incluyendo carcinomas colorrectales, se demostró que era debido a un extremo aguas arriba TCF7L2 elemento de unión a conducir la transcripción de MYC 23,24. Como tal, hay un gran interés en la determinación de los genes aguas abajo regulados por este factor de transcripción clave. Basándose en la experiencia con TCF7L2 como un ejemplo de la metodología, este documento describe cómo generar chip plantilla de ADN de alta calidad. Chip se llevó a cabo en la línea celular de carcinoma colorrectal, HCT116, para la posterior secuenciación para construir una alta resollución mapa de los genes vinculados por TCF7L2 25 en un esfuerzo para producir una mayor comprensión en a su papel clave en la patogénesis de los rasgos complejos.

<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)

Métodos de secuenciación (chip-chip-ss) ofrecen directamente la cobertura de todo el genoma, en donde la combinación de inmunoprecipitación de cromatina (CHIP) y secuenciación masiva en paralelo puede ser utilizado para identificar el repertorio de secuencias de ADN de mamíferos vinculados por factores de transcripción en vivo. &quot;La próxima generación de&quot; tecnologías de secuenciación del genoma proporcionan 1-2 órdenes de magnitud aumento en la cantidad de secuencia que puede ser rentable generado más viejas tecnologías lo que permite métodos de chip-ss para proporcionar directamente la cobertura de todo el genoma de perfiles efectiva de los mamíferos interacciones proteína-DNA. Para exitosos enfoques chip-ss, hay que generar chip molde de ADN de alta calidad para obtener los mejores resultados de secuenciación. La descripción se basa en la experiencia con el producto de proteína del gen más fuertemente implicados en la patogénesis de la diabetes tipo 2, a saber, el factor de transcripción factor de transcripción 7-como 2 (TCF7L2). Este factor también ha sido implicado en varios tipos de cáncer. Contorno es cómo generar plantilla de chip de ADN de alta calidad derivada de la línea celular de carcinoma colorrectal, HCT116, con el fin de construir un mapa de alta resolución a través de la secuenciación para determinar los genes vinculados por TCF7L2, dando más detalles en a su papel clave en la patogénesis de rasgos complejos.

Una vez que la cromatina se ha sonicó y han sido tratados con RNasa y proteinasa, las muestras se ejecutan en el gel de agarosa al 2% deben presentar un frotis con la mayor parte del ADN en el tamaño deseado. Si se prueban varios ciclos diferentes, una disminución gradual en el tamaño debe ser visto como el número de ciclos de aumento (Figura 2). Después de completar la porción de inmunoprecipitación del protocolo el enriquecimiento o bien se puede comprobar por PCR ...

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

La combinación de inmunoprecipitación de la cromatina y la secuenciación de ultra-alto rendimiento (chip-ss) puede identificar y mapear las interacciones proteína-ADN en un tejido o línea celular dada. Contorno es cómo generar una plantilla de chip de alta calidad para la posterior secuenciación, utilizando la experiencia con el factor de transcripción TCF7L2 como un ejemplo.

Generación de Plantilla ADN Immunoprecipitation cromatina alta calidad para la secuenciación de alto rendimiento (chip-ss)

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 vistas.  Children's Hospital of Philadelphia Research Institute. La combinación de inmunoprecipitación de la cromatina y la secuenciación de ultra-alto rendimiento (chip-ss) puede identificar y mapear las interacciones proteína-ADN en un tejido o línea celular dada. Contorno es cómo generar una plantilla de chip de alta calidad para la posterior secuenciación, utilizando la experiencia con el factor de transcripción TCF7L2 como un ejemplo.

Video: 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 los Pequeños ARN moléculas de microARN cuantificación mediante secuenciación de alto rendimiento

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

Investigación

Biología

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.

Dirigida análisis de metilación del ADN mediante secuenciación de próxima generación

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.

Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados

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.

Ensayo de inmunoprecipitación de la cromatina para la Identificación de Arabidopsis proteína-DNA<em&gt; En 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 siguientes: Genoma de toda la cartografía de la proteína-ADN Interacciones de alto rendimiento de secuenciación de fragmentos de ADN adenina-metilado

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.

Amplificación, secuenciación de próxima generación, y Genómica Mapeo de ADN retrovirales sitios de integración

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 rendimiento CRISPR vector de la construcción y caracterización de las modificaciones del ADN mediante la generación de raíces peludas tomate

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.

Un protocolo para la evaluación funcional de la Proteína Total-mutagénesis de saturación Bibliotecas La utilización de secuenciación de alto rendimiento

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.

Ensayo cinético de molécula única de alto rendimiento paralelo para la escoja de ADN específica del sitio

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.

Los métodos modificados para la carga de placas de extracción de ADN de alto rendimiento reducen el potencial de contaminación

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