Damos las gracias a T. Hung, MC. Tsai, O. Manor, E. Segal, M. Kuroda, Swigut T., y yo Shestopalov para los debates. Con el apoyo de la Agencia de Ciencia, Tecnología e Investigación de Singapur (CC), el NIH R01-R01 CA118750 y HG004361-(HYC), y California Instituto de Medicina Regenerativa (HYC). HYC es un científico de Carrera Temprana del Instituto Médico Howard Hughes.

1. Sonda de Diseño Diseño anti-sentido ADN embaldosado sondas para la recuperación selectiva del ARN diana por Chirp. <ol><li> Diseño anti-sentido sondas de oligonucleótidos mediante el diseñador de la sonda en línea en <a href="http://singlemoleculefish.com">singlemoleculefish.com</a> 18. </li><li> Utilice los siguientes parámetros: número de sondas = 1 sonda / 100 pb de longitud ARN, 2) el objetivo% GC = 45, 3) longitud del oligonucleótido = 20, 4) de longitud Espacio = 60-80. Romper el ARN en segmentos, si es demasiado larga para el diseñador. Omitir las regiones de repeticiones o extensa homología. </li><li> Orden anti-sentido sondas de ADN con BiotinTEG a las 3 de alto riesgo extremo. </li><li> Sondas de etiqueta de acuerdo con sus posiciones a lo largo del ARN. Separarlos en dos grupos para que el &quot;par&quot; de piscina contiene todas las sondas de numeración 2, 4, 6, etc, y el &quot;extraño&quot; de piscina contiene sondas de numeración 1, 3, 5, etc Diluir grupo de sondas a la concentración mM 100 y almacenar a -20 ° C. </li><li> Todos los experimentos se realizaron utilizandoambos grupos, que sirven como controles internos para los demás. Real RNA-dependiente de la señal estará presente en las dos piscinas, mientras que la sonda específicos de ruidos será único para cada piscina. Esto se aplica tanto para CHIRP-qPCR y CHIRP ss. </li></ol> 2. Las células de la cosecha Recoger las células que serán usadas en el experimento CHIRP. <ol><li> Se cultivan las células en placas de cultivo de tejidos o frascos a la confluencia. Enjuague con tampón fosfato salino (PBS) de una vez trypsinize. Apaga la tripsina con un volumen de&gt; 2 veces los medios de comunicación, la pipeta hacia arriba y abajo para separar las células y resuspender en suspensión de células individuales. Transfiera todos los medios de comunicación y las células resuspendidas en 50 ml tubos Falcon. 20 millones de células son normalmente suficientes para una muestra Chirp. </li><li> Derivado células en 800RCF durante 4 min. Aspirar y medios Resuspender las células en 40 millones de 40 ml de PBS, se combinan los tubos si es necesario. Derivado células en 800RCF durante 4 min. Decantar PBS, cuidadosamente aspirar en un ángulo del líquido restante. </li></ol&gt; 3. Enlace de la Cruz-Las células y recoger sedimento celular Crosslink recogen las células con glutaraldehído para preservar la cromatina-ARN interacciones y preparar sedimento celular. <ol><li> Siga todos los pasos a temperatura ambiente. </li><li> Preparar 1% de glutaraldehído en PBS temperatura ambiente. Preparar 10 ml por 10 millones de células (0,4 ml de 25% de valores glutaraldehído + 9,6 ml de PBS). El glutaraldehído debe ser utilizado fresco. </li><li> Toque en la parte inferior de los tubos Falcon para desalojar gránulos. Resuspender pellet de células en 1% de glutaraldehído, comenzando con un pequeño volumen para evitar trozos, luego arriba hasta el volumen completo. Invertir para mezclar. Crosslink durante 10 minutos a temperatura ambiente en un agitador de extremo a extremo o rotador. </li><li> Sofocar la reacción de reticulación con el volumen 1/10o de 1,25 M de glicina a temperatura ambiente durante 5 min. </li><li> Girar a 2000RCF durante 5 min. Aspirar el sobrenadante y el precipitado lavado con 20 ml de PBS enfriado una vez, girando a 2000RCF durante 5 min. </li><li> Aspirar y resuspender el wincinera, reticulado precipitado con 1 ml de PBS enfriado por 20 millones de células. Transfiera cada ml a un tubo Eppendorf y giran a 2000RCF durante 3 min a 4 ° C. Quite la mayor cantidad posible con PBS punta de la pipeta con cuidado. </li><li> Flash congelar los gránulos de las células en nitrógeno líquido y se almacena a -80 ° C indefinidamente. </li></ol> 4. Lisis de la célula Lisar células reticuladas para preparar lisado celular. <ol><li> Descongele gránulos congelados de células a temperatura ambiente. Toque duro para desalojar y mezclar el sedimento celular. Girar por la pastilla en el 2000RCF durante 3 min a 4 ° C. Utiliza la esquina 10 l punta de la pipeta para eliminar cualquier resto de PBS. </li><li> En una balanza electrónica (precisión de 1 mg) de tara la masa de un vacío tubo Eppendorf (los tubos pesan 1.060 gramos forma muy consistente). Pese cada pastilla y registrar su peso. Un total de 15 cm plato de células HeLa reticulados normalmente pesa 100 mg. </li><li> Suplemento tampón de lisis (10 veces la masa de sedimento, por ejemplo, 1 ml de 100 mg) con Fresh inhibidor de la proteasa, PMSF y SUPERase en (véase la lista adjunta de búfer). Mezclar bien. </li><li> Añadir un volumen 10 veces complementado tampón de lisis a cada tubo y resuspender el precipitado. Para pequeños gránulos &lt;25 mg, resuspender en 250 l de amortiguación complementado lisis. La suspensión debe ser lisa. Si no es así, dividir la suspensión en 500 ml de alícuotas y utilizar un motor mezclador de pellets para disgregar los grumos. Proceder de inmediato a tratamiento con ultrasonidos. </li></ol> 5. Sonicación ADN cizallamiento por sonicación reticuladas lisados ​​celulares. <ol><li> Someter a ultrasonidos lisado celular en Bioruptor en 15 ml tubos Falcon. Utilice &lt;1,5 ml de lisado en cada tubo, y para acelerar la sonicación, sonicar no más de dos tubos a la vez. </li><li> Someter a ultrasonidos en un baño de agua a 4 ° C en la posición más alta con 30 segundos en ON, OFF 45 segundos intervalos de pulso. Compruebe lisado cada 30 minutos. Continuar sonicando hasta que el lisado de células ya no se turbia. Esto puede tomar tan poco como 30 minutos y horas hasta 4. El númerode los tubos, el volumen de la muestra, la temperatura del baño, y el período de tiempo de sonicación afectará cuánto tiempo toma el proceso. Tubos probablemente sonicar a un ritmo diferente, así que la piscina juntos cada 30 minutos y redistribuir en los tubos originales para asegurar la homogeneidad. Nota: glutaraldehído reticulado células toman mucho más tiempo para sonicar que los equivalentes de formaldehído. </li><li> Al lisado se vuelve clara, transferir 5 l lisado a un tubo Eppendorf. Añadir 90 l de ADN de la proteasa K (PK) Buffer (véase la lista de amortiguación) y 5 PK l. Vortex para mezclar y centrifugar brevemente. Incubar durante 45 min a 50 ° C. </li><li> Extraer el ADN con el kit de Qiagen purificación de PCR. ADN se eluye en 30 l del buffer de elución Qiagen (EB) y comprobar el tamaño del ADN, el 1% en gel de agarosa. Si el frotis grueso de ADN es 100-500 pb, sonicación es completa. Si no es así, continúe con ultrasonidos. </li><li> Centrifugar las muestras de ultrasonidos a 16100RCF durante 10 min a 4 ° C. Combine sobrenadantes, alícuotas en muestras de 1 ml y flash de congelación en nitroge líquidon. Conservar a -80 º C. </li></ol> 6. CHIRP Hibridar sondas biotiniladas de ADN a ARN y aislar la cromatina enlazado. <ol><li> Descongele los tubos de la cromatina a temperatura ambiente. </li><li> Prepare el tampón de hibridación (véase la lista de buffers, preparar 2 ml por cada ml de la cromatina). Vortex para mezclar. </li><li> Para una muestra típica CHIRP utilizando 1 ml de lisado, quitar 10 l para INPUT ARN y 10 l de entrada de ADN y el lugar en tubos Eppendorf. Mantener en hielo hasta su uso posterior. </li><li> Transferir 1 ml de la cromatina a 15 ml tubo Falcon. Añadir 2 ml tampón de hibridación a cada tubo. Por volumen total &lt;1,5 ml, utilice tubos Eppendorf. </li><li> Descongele sondas a temperatura ambiente. NanoDrop sondas para comprobar la cantidad si no lo ha usado en mucho tiempo (100 uM sondas deberían especificaciones ~ 500-600 ng / l usando el ajuste de una sola cadena de ADN). Añadir un volumen adecuado de sondas específicas para tubos (100 pmol por sonda 1 ml de la cromatina, 1 l de 100 pmol / l sonda por 1 ml de la cromatina).Mezclar bien. Incubar a 37 ° C durante 4 horas con agitación. </li><li> Con 20 minutos restantes para la hibridación, preparar las perlas magnéticas C-1 (almacenado a 4 ° C). Utilizar 100 l por 100 pmol de las sondas. Lavar con 1 ml de tampón de lisis sin suplementos tres veces, usando la cinta magnética Dynamag-2 a cuentas separadas de búfer. </li><li> Resuspender las perlas en el volumen original de tampón de lisis, y Suplemento con fresco PMSF, PI y SUPERase en-. Después de 4 h reacción de hibridación es completa, añadir 100 l perlas a cada tubo. Mezclar bien. Incubar a 37 ° C durante 30 min con agitación. </li><li> Preparar el tampón de lavado (5 ml por muestra). Vortex para mezclar. Pre-caliente a 37 ° C. Añadir PMSF antes de su uso. </li><li> Lávese las cuentas con 1 ml de solución de lavado cinco veces. En el primer lavado, utilizar Dynamag-15 banda magnética a perlas separadas, decantación y se resuspenden en 1 ml de tampón de lavado. Transferir el volumen a 1,5 ml tubo Eppendorf. Incubar a 37 ° C con agitación durante 5 min. </li><li> En lavados posteriores, la desaceleración de cada tubo en un minicentrifuge, Ajuste de la muestra en Dynamag-2 banda magnética durante 1 min. Se decanta la muestra, limpie cualquier derrame con un Kimwipe, resuspender en 1 ml de solución de lavado. Incubar a 37 ° C con agitación durante 5 min. Repetir durante cinco lavados totales. </li><li> En el último lavado, resuspender las cuentas bien. Sacar 100 l, y reservado para el aislamiento de ARN. Reserva de 900 l para la fracción de ADN. Coloque todos los tubos de Dynamag-2 la banda magnética y quitar el tampón de lavado. Girar todos los tubos durante un breve instante, colocarlos en cinta magnética. Eliminar el último bit de buffer de lavado completamente con una fuerte punta de la pipeta 10 l. </li></ol> 7. Aislamiento del ARN Extracto de la fracción de ARN de las muestras de un pitido para cuantificar de QRT-PCR. <ol><li> Tomar muestras de 100 l de talón y 10 l de muestra ENTRADA ARN. Añadir 85 l de ARN PK buffer de pH 7,0 a ARN de entrada. Volver a suspender las bolas en 95 l de pH 7,0 ARN PK búfer. Añadir 5 K Proteinease l e incubar a 50 ° C durante 45 min con agitación de extremo a extremo. </li><li> En pocas palabras de girar todos los tubos yhervir las muestras durante 10 minutos en el bloque de calor a 95 ° C. </li><li> Enfriar en hielo, añadir 500 l TRIzol vórtice, vigorosamente durante 10 segundos. Incubar a temperatura ambiente durante 10 min. Conservar a -80 ° C o vaya al paso 4. </li><li> Añadir 100 l de cloroformo TRIzol muestras tratadas. Vortex vigorosamente durante 10 segundos. Girar a 16100RCF en una centrífuga de mesa durante 15 min a 4 ° C. </li><li> Retire ~ 400 l sobrenadante acuoso, evitando orgánica y la interfaz. </li><li> Añadir 600 l (1,5 volúmenes) 100% de etanol y mezclar bien. Haga girar la muestra a través MIRNeasy mini columnas. Lavar 1x con TAR (MIRNeasy mini kit), 2x con RPE de acuerdo al protocolo del fabricante. Eluir con 30 l de nucleasa libre de H 2 O (HNF 2 O). </li><li> Tratar el eluato de ARN con ADN libre por el protocolo del fabricante. Después de la reacción es completa, calentar la muestra durante 15 minutos a 65 ° C para inactivar completamente cualquier resto de DNasa. </li><li> Utilice 1 l de ARN aislado por pocillo de QRT-PCR análisis para confirmar lncRNArecuperación. GAPDH se utiliza a menudo como un control negativo. </li></ol> 8. El aislamiento del ADN Extracto de la fracción de ADN de muestras de un pitido para identificar o cuantificar mediante secuenciación por PCR cuantitativa. <ol><li> Prepare el tampón de elución del ADN (ver lista de buffers), 150 l por ejemplo, incluyendo la entrada de ADN. </li><li> Añadir 1 0μL RNasa A (10 mg / ml) y 10 l RNasa H (10 U / l) por ml de tampón de elución del ADN, y vórtice para mezclar. </li><li> Resuspender cada muestra de las perlas en 150 l de tampón de elución con ADN RNasas. (ENTRADA Resuspender el ADN en 140 l) Se incuba a 37 ° C durante 30 min con agitación. </li><li> Cuentas separadas y el sobrenadante de Dynamag-2 la banda magnética. Eliminar el sobrenadante y añadir a los tubos etiquetados. </li><li> Preparar una segunda alícuota de tampón de elución de ADN con 10 l de ribonucleasa A (10 mg / ml) y RNasaH (10 U / l) exactamente como se hace en 8,2). Añadir 150 l de cada muestra (incluida la entrada de ADN), se incuba, y eliminar el sobrenadante. Recoge todas sobrenadante (hombrod es ~ 300 l). </li><li> Añadir 15 PK l de cada muestra. Incubar a 50 ° C durante 45 min con agitación. </li><li> Pre-spin hacia abajo de color amarillo fase de bloqueo de tubos de gel (5PRIME). Transferir las muestras de ADN a los tubos de gel de enganche de fase, y añadir 300 l PhOH: cloroformo: isoamílico por muestra. Agitar enérgicamente durante 10 minutos, y de girar en una centrífuga de mesa a 16100RCF durante 5 min a 4 ° C. Tome acuoso de la parte superior (~ 300 l). Añadir 3 GlycoBlue l, 30 l de NaOAc y 900 l de EtOH 100%. Mezclar bien y guardar a -20 ° C durante la noche. </li><li> Haga girar las muestras a 16100RCF durante 30 min a 4 ° C. </li><li> Decantar el sobrenadante con cuidado. Añadir 1 ml de EtOH 70% y agitar para mezclar. Girar a 16100RCF durante 5 min. Eliminar sobrenadante con una pipeta. Deje secar al aire durante 1 min. Resuspender en 30 l EB. </li><li> Las muestras de ADN están listos para el análisis de qPCR o la preparación de las bibliotecas de secuenciación de alto rendimiento por el protocolo de Illumina. </li></ol> 10. Los resultados representativos Figura 1 </strong&gt; representa el flujo de trabajo CHIRP. Un experimento CHIRP éxito típicamente enriquece ARN diana significativamente más ARNs no específicos. Figura 2 muestra el enriquecimiento de la telomerasa humana ARN (TERC) a partir de células HeLa por GAPDH, un ARN abundantes celulares que sirve como un control negativo. La mayoría de los ARNs TERC (~ 88%) presentes en la célula se empujado hacia abajo por la realización de chirrido, mientras que sólo el 0,46% de GAPDH RNA fue recuperada, demostrando un factor de enriquecimiento de ~ 200 veces. Las sondas no específicas, tales como sondas dirigidas LacZ ARN, que no se expresa en células de mamíferos (Figura 2), se puede utilizar como controles negativos adicionales. Regiones de ADN esperados de obligar a la lncRNA objetivo son típicamente enriquecida sobre regiones negativos cuando se mide por qPCR. Figura 3 muestra la validación qPCR de cuatro AIRE CALIENTE enlazados a sitios en fibroblastos primarios de prepucio humano que determina realizando CHIRP-ss en la misma línea celular, mientras que TERC y GAPDH ADN sitios sírve como regiones de control negativo. Tanto el &quot;par&quot; y la sonda &quot;raro&quot;, establece el enriquecimiento producido comparable de plazos previstos AIRE CALIENTE-sitios a través de las regiones negativas, un sello distintivo de los verdaderos lncRNA sitios de unión. De alto rendimiento de secuenciación de ADN CHIRP enriquecido se obtiene un mapa global de los sitios de unión lncRNA. La Drosophila lncRNA roX2 se sabe que interactúan con el cromosoma X en una forma que se requiere para la compensación de la dosis. La Figura 4 muestra roX2 perfil de unión sobre una sección del cromosoma X. Tanto &quot;incluso&quot; y &quot;extraño&quot; las muestras han sido secuenciados y sus únicos ruidos han sido eliminados para producir un registro de las señales superpuestas. Cada &quot;pico&quot; aquí indica un sitio fuerte de roX2 vinculante. La pista completa y la lista de roX2 genes diana se han descrito en Chu et al. 2011 17. <img alt="Figura 1." src="/files/ftp_upload/3912/3912fig1.jpg" /> Figura 1. Diagrama de flujo del procedimiento CHIRPDuré. La cromatina se entrecruza con lncRNA: aductos de proteínas in vivo Biotinylated sondas suelo de baldosas se hibridan para apuntar lncRNA, y los complejos de la cromatina se purifica mediante perlas magnéticas estreptavidina, seguido de lavados rigurosos.. Nos eluir el ADN o las proteínas lncRNA unido con un cóctel de RNasa A y H. se esquematiza una supuesta secuencia de lncRNA vinculante en color naranja. Anteriormente publicado en Chu et al. 2011. 17 <img alt="Figura 2." src="/files/ftp_upload/3912/3912fig2.jpg" /> Figura 2. CHIRP enriquece de ARN humano TERC. TERC-asDNA sondas recuperar ~ 88% de los celulares TERC RNA no detectable y GAPDH. LacZ asDNA sondas se utilizaron como controles negativos y recuperar ni ARN. La media ± desviación estándar se muestran. Anteriormente publicado en Chu et al. 2011. 17 <img alt="Figura 3." src="/files/ftp_upload/3912/3912fig3.jpg" /> Figura 3. AIRE CALIENTE CHIRP-qPCR en humanos primariafibroblastos Eskin. NFKBIA, HOXD3 4, SERINC5 y ABCA2 son las regiones que interactúan con AIRE CALIENTE. TERC y GAPDH sirvieron como controles negativos. La media ± desviación estándar se muestran. Anteriormente publicado en Chu et al. 2011. 17 <img alt="Figura 4." src="/files/ftp_upload/3912/3912fig4.jpg" /> Figura 4. CHIRP-ss datos de roX2 ARN en células de Drosophila SL2. &quot;Aún&quot; y &quot;extraño&quot; fueron secuenciados por separado, sus datos se unen para reflejar sólo los picos comunes en ambos. La pista fusionada se muestra. Anteriormente publicado en Chu et al. 2011. 17

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Maps+of+Long+Noncoding+RNA+Occupancy+Reveal+Principles+of+RNA-Chromatin+Interactions.">Genomic Maps of Long Noncoding RNA Occupancy Reveal Principles of RNA-Chromatin Interactions.</a> Mol. Cell. , (2011).</li><li>Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+individual+mRNA+molecules+using+multiple+singly+labeled+probes.">Imaging individual mRNA molecules using multiple singly labeled probes.</a> Nat. Methods. 5, 877-879 (2008).</li></ol>

C. Chu y Chang HY se nombran como inventores en una solicitud de patente basada en este método.

Aquí se describe CHIRP-ss, un método de asignación en vivo sitios lncRNA vinculantes en todo el genoma. Los parámetros clave para el éxito son las piscinas abiertas de mosaico sondas de oligonucleótidos y el entrecruzamiento glutaraldehído. El diseño de las sondas de afinidad es sencillo dada la secuencia de ARN y no requiere conocimiento previo de la estructura del ARN o dominios funcionales. Nuestro éxito con roX2, TERC, y AIRE CALIENTE - tres ARN en lugar de dos especies diferentes - sugiere que CHIRP-ss es probable generalizable a muchos lncRNAs. Al igual que con todos los experimentos, los controles de asistencia y adecuada están obligados a interpretar los resultados. LncRNA diferentes pueden requerir un ajuste de las condiciones, y el cambio razonable de condiciones, como la selección de sondas de afinidad o reticulantes diferentes, puede poner de relieve diferentes aspectos de las interacciones ARN-cromatina. Al igual que el chip-ss, no todos los eventos son necesariamente vinculantes funcional, y se requieren estudios adicionales para determinar las consecuencias biológicas de ARN occupancy en la cromatina. No obstante, prevemos muchas aplicaciones interesantes de esta tecnología para los investigadores de otras asociadas a la cromatina lncRNAs, que ahora se cuentan por miles 8,9. Así como chip-ss ha abierto la puerta a todo el genoma de la exploración de las interacciones proteína-ADN, chirrido-ss estudios de la &quot;interactoma ARN&quot; puede revelar muchas nuevas vías de la biología.

Buffer List:
Dissolve a pellet of complete protease inhibitor in 1 ml water as 50x stock. Make 100 mM PMSF in isopropanol (100x stock). Superase-in is used as 200x stock. Store all at -20&deg;C.
Lysis Buffer:
50 mM Tris-Cl pH 7.0 
10 mM EDTA 
1% SDS 
Always add PMSF, P.I. and Superase-in fresh before use except when washing beads
Proteinase K Buffer (for DNA)
100 mM NaCl 
10 mM TrisCl pH 8.0 (For RNA use pH 7.0) 
1 mM EDTA 
0.5% SDS 
Add 5% by volume Proteainse K (Ambion AM2546 20 mg/ml) fresh before use
Hybridization Buffer
750 mM NaCl 
1% SDS 
50 mM Tris-Cl pH 7.0 
1 mM EDTA 
15% formamide (store in the dark at 4&deg;C) 
Always add PMSF, P.I. and Superase-in fresh before use
Wash Buffer
2x NaCl and Sodium citrate (SSC) (diluted from 20x SSC Invitrogen stock) 
0.5% SDS 
Always add PMSF fresh before use
DNA elution Buffer
50 mM NaHCO3 
1% SDS
Table of specific reagents and equipment:
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (EM grade)</td>
			<td>Sigma Aldrich</td>
			<td>G5882-10x10ml</td>
		</tr>
		<tr>
			<td>Motorized pellet mixer</td>
			<td>VWR</td>
			<td>V8185-904</td>
		</tr>
		<tr>
			<td>Protease inhibitor</td>
			<td>Roche</td>
			<td>11873580001</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma Aldrich</td>
			<td>78830</td>
		</tr>
		<tr>
			<td>Superase-in</td>
			<td>Ambion</td>
			<td><a href="http://www.ambion.com/catalog/CatNum.php?AM2696">AM2696</a></td>
		</tr>
		<tr>
			<td>Bioruptor</td>
			<td>Diagenode</td>
			<td>UCD-200</td>
		</tr>
		<tr>
			<td>Falcon tubes (for sonication)</td>
			<td>Corning</td>
			<td>430790</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Ambion</td>
			<td>AM2546</td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28106</td>
		</tr>
		<tr>
			<td>C-1 magnetic beads</td>
			<td>Invitrogen</td>
			<td>65002</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma Aldrich</td>
			<td>P7626-25G</td>
		</tr>
		<tr>
			<td>DynaMag-15 magnet</td>
			<td>Invitrogen</td>
			<td>123-01D</td>
		</tr>
		<tr>
			<td>DynaMag-2 magnet</td>
			<td>Invitrogen</td>
			<td>123-21D</td>
		</tr>
		<tr>
			<td>MIRNeasy mini kit</td>
			<td>Qiagen</td>
			<td>217004</td>
		</tr>
		<tr>
			<td>Rnase H</td>
			<td>Epicentre</td>
			<td>R0601K</td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Sigma Aldrich</td>
			<td>R4875-100MG</td>
		</tr>
		<tr>
			<td>Phase Lock Gel Heavy</td>
			<td>5 PRIME</td>
			<td>2302810</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
		</tr>
		<tr>
			<td>Phenol:chloroform:Isoamyl</td>
			<td>Invitrogen</td>
			<td>15593-031</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Ricca</td>
			<td>RSOC0020-1C</td>
		</tr>
		<tr>
			<td>GlycoBlue</td>
			<td>Ambion</td>
			<td>AM9515</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>J.T. Baker</td>
			<td>4057-06</td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Invitrogen</td>
			<td>10010-049</td>
		</tr>
		<tr>
			<td>Elution Buffer (EB)</td>
			<td>Qiagen</td>
			<td>19086</td>
		</tr>
		<tr>
			<td>20x SSC</td>
			<td>Invitrogen</td>
			<td>15557-036</td>
		</tr>
		<tr>
			<td>10% SDS</td>
			<td>Invitrogen</td>
			<td>15553-027</td>
		</tr>
		<tr>
			<td>DNA-free</td>
			<td>Ambion</td>
			<td>AM1906</td>
		</tr>
		<tr>
			<td>Buffer kit</td>
			<td>Ambion</td>
			<td>AM9010</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Invitrogen</td>
			<td>15515-026</td>
		</tr>
	</tbody>
</table>

chromatin isolation by rna purification (chirp)

RNAs no codificantes largos son los principales reguladores de los estados de la cromatina de importantes procesos biológicos como la compensación de la dosis, la impresión y la expresión de genes en el desarrollo 1,2,3,4,5,6,7. El reciente descubrimiento de miles de lncRNAs en asociación con determinados complejos de la cromatina modificación, tales como el complejo Polycomb represiva 2 (PRC2) que media la histona H3 lisina 27 trimethylation (H3K27me3), sugiere que las funciones generales de lncRNAs numerosos estados en la gestión de la cromatina en un gen específico 8,9 de la moda. Mientras que algunos lncRNAs se cree que actúa en cis de los genes vecinos, lncRNAs otros trabajan en trans para regular los genes yacimiento lejano. Por ejemplo, Drosophila lncRNAs roX1 roX2 y regiones se unen numerosos en el cromosoma X de las células masculinas, y son fundamentales para la compensación de la dosis 10,11. Sin embargo, la ubicación exacta de sus sitios de unión no se conocen en alta resolución. Del mismo modo, humana lncRNA AIRE CALIENTE puede afectar a la ocupación de PRC2 hientos de los genes en todo el genoma 3,12,13, pero ¿cómo se logra la especificidad no está claro. LncRNAs también puede servir como andamios modulares para contratar el montaje de complejos de proteínas múltiples. El clásico de acción trans ARN andamio es el TERC ARN que sirve como la plantilla y andamio para la telomerasa complejo 14; AIRE CALIENTE también puede servir como un andamio para PRC2 y un desmetilasa H3K4 complejo 13. Los estudios previos de cartografía de ocupación del ARN en la cromatina han revelado una sólida perspectiva de 15,16, pero sólo en un lugar único gen a la vez. Los sitios de ocupación de la mayoría de lncRNAs no se conocen, y las funciones de regulación de la cromatina en lncRNAs han sido en su mayoría infiere de los efectos indirectos de la perturbación lncRNA. Al igual que la cromatina immunoprecipitation seguida de microarrays o la secuenciación profunda (chip-chip o chip siguientes, respectivamente) ha mejorado enormemente nuestra comprensión de las interacciones proteína-ADN en una escala genómica, aquí se ilustra una recentemente publicada estrategia para asignar tiempo de ocupación en todo el genoma ARN en alta resolución 17. Este método, Aislamiento cromatina por purificación del RNA (CHIRP) (Figura 1), se basa en la captura de afinidad de destino lncRNA: complejo cromatina por embaldosado antisentido-oligos, que luego se genera un mapa de sitios de unión genómicas a una resolución de varios cientos de bases con sensibilidad alta y baja de fondo. Chirp es aplicable a muchos lncRNAs porque el diseño de sondas de afinidad es sencillo dada la secuencia de ARN y no requiere conocimiento de la estructura del ARN o dominios funcionales.

Watch this Scientific Journal Video about Chromatin Isolation by RNA Purification ChIRP at JoVE.com

Chromatin Isolation by RNA Purification ChIRP

Chirp es una técnica novedosa y rápida a los sitios de unión del Mapa del Genoma de los ARNs no codificantes de largo (lncRNAs). El método tiene la ventaja de la especificidad de los oligonucleótidos antisentido baldosas para permitir la enumeración de los sitios genómicos lncRNA enlazados.

Aislamiento de la cromatina mediante la purificación de ARN (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

chromatin-isolation-by-rna-purification-chirp

Research

JoVE Journal

Biology

Chromatin Isolation by RNA Purification (ChIRP)

85.7K vistas.  Stanford University School of Medicine. Chirp es una técnica novedosa y rápida a los sitios de unión del Mapa del Genoma de los ARNs no codificantes de largo (lncRNAs). El método tiene la ventaja de la especificidad de los oligonucleótidos antisentido baldosas para permitir la enumeración de los sitios genómicos lncRNA enlazados.

Video: Chromatin Isolation by RNA Purification ChIRP

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Investigación

Biología

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Aislamiento y purificación de Drosophila Clasificación de las neuronas periféricas por bolas magnéticas

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Generación de ADN / ARN híbridos en el ADN genómico de transformación utilizando oligonucleótidos de ARN que contiene

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

Aislamiento y caracterización de ARN que contienen exosomas

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

La endocitosis en vivo de hígado seguido por la purificación de las células hepáticas de la perfusión del hígado

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Aislamiento y purificación de Kinesin desde Drosophila Los embriones

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

En la transcripción in vitro y la nivelación de mRNA de luciferasa Gaussia Seguido por transfección de células HeLa

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Aislamiento rápido y purificación de mitocondrias para el trasplante por disociación de tejidos y filtración diferencial

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

Generación y purificación de Humanos INO80 remodelación de la cromatina complejos y Subcomplejos

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Aislamiento de ARN de ratón Páncreas: Un tejido de ribonucleasa-rica

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...