iClip - transcriptoma en todo el mapeo de la proteína-ARN interacciones con la Resolución de nucleótidos individuales

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Summary

La disposición espacial de ARN-proteínas de unión en una transcripción es un determinante clave de la regulación post-transcripcional. Por lo tanto, hemos desarrollado cada nucleótido reticulación UV resolución y inmunoprecipitación (iClip) que permite que precisa todo el genoma de mapeo de los sitios de unión de una proteína de unión al ARN.

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Konig, J., Zarnack, K., Rot, G., Curk, T., Kayikci, M., Zupan, B., Turner, D. J., Luscombe, N. M., Ule, J. iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution. J. Vis. Exp. (50), e2638, doi:10.3791/2638 (2011).

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Abstract

La composición y disposición espacial de los ARN-proteínas de unión (prácticas comerciales restrictivas) en una transcripción guía de los diversos aspectos de la regulación post-transcripcional 1. Por lo tanto, un paso esencial hacia la comprensión de la regulación transcripción a nivel molecular consiste en obtener información de posición en los sitios de unión de las prácticas comerciales restrictivas 2.

Las interacciones proteína-ARN puede ser estudiado con métodos bioquímicos, pero estos métodos no se ocupan de unión al ARN en su contexto celular de origen. Los primeros intentos de estudiar el ARN-proteína complejos en su entorno celular empleado purificación por afinidad o inmunoprecipitación combinadas con pantalla diferencial o de análisis de microarrays (RIP-CHIP) 3-5. Estos enfoques son propensos a la identificación de interacciones directas e indirectas o no fisiológico-6. Con el fin de aumentar la especificidad y la resolución de posición, una estrategia conocida como CLIP (UV entrecruzamiento e inmunoprecipitación) se introdujo 7,8. SEGMENTO combina UV entrecruzamiento de las proteínas y las moléculas de ARN con esquemas de purificación riguroso incluyendo electroforesis en gel de poliacrilamida desnaturalizante. En combinación con las tecnologías de secuenciación de alto rendimiento, CLIP ha demostrado ser una poderosa herramienta para estudiar las interacciones proteína-ARN en un genoma de gran escala (en adelante, HITS-CLIP o CLIP-ss) 9,10. Recientemente, PAR-CLIP se introdujo que utiliza análogos fotorreactivos ribonucleósido para la reticulación 11,12.

A pesar de la alta especificidad de los datos obtenidos, los experimentos clip suelen generar bibliotecas de cDNA de la secuencia de complejidad limitada. Esto es en parte debido a la cantidad limitada de co-ARN purificado y las dos reacciones ineficientes ligadura de ARN necesario para la preparación de la biblioteca. Además, los ensayos de extensión del cebador indicó que muchos ADNc truncado prematuramente en el reticulado de nucleótidos 13. Tal ADNc truncado se pierden durante el protocolo de preparación del clip de la biblioteca estándar. Recientemente hemos desarrollado iClip (individual-nucleótidos clip de resolución), que captura el ADNc truncado mediante la sustitución de uno de los pasos intermolecular ineficiente ligadura de ARN con una más eficiente circularización intramolecular cDNA (Figura 1) 14. Es importante destacar que la secuenciación del ADNc truncado proporciona información detallada sobre la posición del sitio de enlace cruzado con una resolución de nucleótidos. Hemos aplicado con éxito para estudiar iClip hnRNP organización de partículas C en un genoma de gran escala y evaluar su papel en la regulación de empalme 14.

Protocol

1. UV entrecruzamiento de las células de cultivo de tejidos

  1. Retire el papel y añadir 6 ml de helado de PBS a las células cultivadas en una placa de 10 cm (suficiente para tres experimentos).
  2. Retire la tapa y colocar en hielo. Irradiar una vez con 150 mJ / cm 2 a 254 nm.
  3. Recoger las células mediante el raspado con una grúa móvil.
  4. Transferencia de 2 ml de suspensión celular a cada uno de tres microtubos. Giran a velocidad máxima durante 10 segundos a 4 ° C para precipitar las células, a continuación, retire el sobrenadante.
  5. Complemento congelar los gránulos de las células en hielo seco y almacenar a -80 ° C hasta su uso.

2. Bolas de preparación

  1. Añadir 100 ml de la proteína A Dynabeads (Dynal, 100.02) por cada experimento para un microtubo nuevo (uso de la proteína G Dynabeads de un ratón o anticuerpos de cabra).
  2. Lávese las cuentas de 2x con buffer de lisis (50 mM Tris-HCl, pH 7,4, 100 mM NaCl, 1% NP-40, 0,1% SDS, 0,5% desoxicolato de sodio, 1 / 100 inhibidor de la proteasa cóctel III, Calbiochem).
  3. Volver a suspender las bolas en 100 l de tampón de lisis con el anticuerpo 2.10 mg.
  4. Gire los tubos a temperatura ambiente durante 30-60 min.
  5. Lave 3 veces con 900 l de buffer de lisis y dejar en el último lavado, hasta que esté listo para continuar con el paso 4.1.

3. Lisis celular y la digestión parcial del ARN

  1. Resuspender el botón celular en 1 ml de solución amortiguadora de lisis y la transferencia de 1,5 ml microtubos.
  2. Prepare una dilución de 1 / 500 de RNasa I (Ambion, AM2295). Añadir 10 l de RNasa dilución I, así como 2 l Turbo DNasa para el lisado celular (1 / 500 diluciones RNasa I [bajo RNasa] se utilizan para la preparación de la biblioteca, 1 / 50 diluciones [alta RNasa] son ​​necesarios para el control de la especificidad de anticuerpos) .
  3. Incubar las muestras de exactamente 3 minutos a 37 ° C, agitando a 1100 rpm. Transferir inmediatamente a hielo.
  4. Girar a 4 ° C y 22.000 g durante 20 minutos para eliminar el lisado. Recoger cuidadosamente el sobrenadante (dejar unos 50 l de lisado con la pastilla).

4. Inmunoprecipitación

  1. Quitar el tampón de lavado de los granos (a partir del paso 2.5), a continuación, añadir el lisado celular (del paso 3.4).
  2. Girar las muestras durante 2 horas a 4 ° C.
  3. Descartar el sobrenadante y lavar los granos con 2x 900 l alto contenido de sal buffer (50 mM Tris-HCl, pH 7,4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0,1% SDS, 0,5% desoxicolato de sodio).
  4. Lavar 2 veces con 900 l de tampón de lavado (20 mM Tris-HCl, pH 7,4, 10 mM MgCl 2, 0,2% Tween-20).

5. Desfosforilación de 3'ends ARN

  1. Descartar el sobrenadante y resuspender las cuentas en 20 l de mezcla PNK (15 l de agua, 4 pH 6,5 l 5x PNK buffer [350mMTris-HCl, pH 6,5; 50mMMgCl 25mMdithiothreitol 2], 0,5 l enzima PNK, 0,5 l RNasin [Promega]).
  2. Incubar durante 20 min a 37 ° C.
  3. Añadir 500 l de tampón de lavado y lavado 1X con alto contenido de sal de amortiguación.
  4. Lavar 2 veces con tampón de lavado.

6. La ligadura de enlazador para ARN 3 '

  1. Retire con cuidado el sobrenadante y resuspender las cuentas en 20 l mezcla de ligación (9 l de agua; 4 buffer l ligadura de 4x [200 mMTris-HCl; MM GCL 40m 2, 40 mM ditiotreitol], 1 l de ARN ligasa [ORC], 0,5 l RNasin [Promega], 1,5 l de pre-adenilado enlazador L3 [20 m]; 4 l PEG400 [81170, Sigma]).
  2. Incubar toda la noche a 16 ° C.
  3. Añadir 500 l de tampón de lavado y lavar 2 veces con 1 ml de buffer de alto contenido de sal.
  4. Lavar 2 veces con 1 ml de solución de lavado y dejar en 1 ml del segundo lavado.

7. Etiquetado de ARN extremo 5 '

  1. Eliminar el sobrenadante y resuspender las bolas en 8 l de mezcla en caliente PNK (0,4 l PNK [ORC], 0,8 l 32 P-γ-ATP, 0,8 l de amortiguación 10 veces PNK [ORC], 6 l de agua).
  2. Incubar durante 5 min a 37 ° C.
  3. Retire la mezcla PNK caliente y volver a suspender las bolas en 20 l de tampón de carga 1x NuPAGE (Invitrogen).
  4. Incubar en un termomezclador a 70 ° C durante 10 min.
  5. Coloque inmediatamente en un imán para precipitar las cuentas vacías y cargar el sobrenadante en el gel (vea el paso 8).

8. SDS-PAGE y la transferencia de la membrana

  1. Cargar las muestras en un NuPAGE 4-12% Bis-Tris gel (Invitrogen) de acuerdo con las instrucciones del fabricante. Use 0,5 l de 1x MOPS funcionamiento de amortiguación (Invitrogen). También carga de 5 l de un marcador de proteína pre-teñido de tamaño (por ejemplo: página gobernante más, Fermentas, SM1811).
  2. Dejar correr el gel durante 50 minutos a 180 V.
  3. Quitar la parte frontal de gel y desechar los residuos sólidos (libre contiene ATP radiactivo).
  4. La transferencia de los complejos ARN-proteína del gel a una membrana de nitrocelulosa utilizando el aparato de transferencia de Novex húmeda de acuerdo con las instrucciones del fabricante (Invitrogen, la transferencia de 1 hora a 30 V).
  5. Después de la transferencia, lavar la membrana en tampón PBS, luego se envuelve en el abrigo de saran y exponerla a una película Fuji a -80 ° C (coloque una pegatina fluorescente al lado de la membrana para alinear más tarde tque el cine y la membrana, realizar exposiciones durante 30 minutos, 1 hora y durante la noche).

9. Aislamiento de ARN

  1. Aislar los complejos ARN-proteína en el experimento bajo RNasa utilizando la autorradiografía del paso 8.5 como una máscara. Cortar este trozo de la membrana en varias rodajas y colocarlas en un microtubo de 1.5 ml.
  2. Añadir 200 l PK buffer (100 mM Tris-HCl pH 7,4, 50 mM NaCl, 10 mM EDTA) y 10 l de proteinasa K (Roche, 03115828001) a las piezas de la membrana. Incubar con agitación a 1100 rpm durante 20 minutos a 37 ° C.
  3. Añadir 200 ul de buffer PKurea (100 mM Tris-HCl pH 7,4, 50 mM NaCl, 10 mM EDTA, 7 M urea) y se incuba durante 20 min a 37 ° C.
  4. Recoger la solución y añadir junto con 400 l de fenol / cloroformo (Ambion, 9722) a un tubo de 2 ml de fase de bloqueo Gel pesados ​​(713 a 2.536, VWR) de ARN.
  5. Incubar durante 5 min a 30 ° C, agitando a 1100 rpm. Separar las fases girando durante 5 minutos a 13.000 rpm a temperatura ambiente.
  6. La transferencia de la capa acuosa a un tubo nuevo (con cuidado de no tocar el gel con la pipeta). Añadir 0,5 l glycoblue (Ambion, 9510) y 40 l de sodio 3 M pH 5,5 acetato y mezclar. Luego, agregue 1 ml de etanol al 100%, mezcla de nuevo y se precipitan por la noche a -20 ° C.

10. La transcripción inversa

  1. Girar durante 20 minutos a 15.000 rpm y 4 ° C. Eliminar el sobrenadante y lavar el precipitado con 0,5 ml de etanol al 80%.
  2. Resuspender el precipitado en 7,25 l de ARN / mezcla de cebadores (6,25 l de agua, 0,5 imprimación Rclip l [0.5pmol/μl], 0,5 l de mezcla dNTP [10 mM]). Para cada experimento o reproducir, utilizar una imprimación Rclip diferentes que contienen las secuencias individuales de código de barras (ver 14).
  3. Incubar durante 5 min a 70 ° C antes de enfriar a 25 ° C.
  4. Añadir 2,75 l mezcla RT (2 l 5x buffer RT, 0,5 l 0,1 M TDT, 0,25 l Superíndice III de la transcriptasa inversa [Invitrogen]).
  5. Incubar 5 min a 25 ° C, 20 min a 42 ° C, 40 min a 50 ° C y 5 min a 80 ° C antes de la refrigeración a 4 ° C.
  6. Añadir 90 l de buffer TE, 0,5 l glycoblue y 10 l de acetato de sodio pH 5,5 y mezclar. A continuación, añadir 250 l de etanol 100%, mezcla de nuevo y se precipitan por la noche a -20 ° C.

11. Gel de purificación de cDNA

  1. Decantar y lavar las muestras (véase 10.1), a continuación, volver a suspender las pastillas en 6 l de agua.
  2. Añadir 6 l 2x TBE-urea tampón de carga (Invitrogen). Muestras de calor a 80 ° C durante 3 minutos antes de cargar directamente.
  3. Cargar las muestras en un 6% prefabricado TBE-urea gel (Invitrogen) y una duración de 40 minutos a 180 V como se describe por el fabricante. También carga un marcador de bajo peso molecular para el corte posterior (ver más abajo).
  4. Corte tres bandas en 120-200 nt (alto), 85-120 nt (medio) y 70-85 nt (bajo). Use theupper tinte y las marcas en el soporte de gel de plástico para guiar a la escisión (ver Figura 3). Tenga en cuenta que el primer Rclip y la secuencia de L3 en conjunto representan el 52 nt de la secuencia de CLIP.
  5. Añadir 400 l de TE y aplastar la rebanada de gel en pequeños trozos con un émbolo de jeringa de 1 ml. Incubar con agitación a 1100 rpm durante 2 horas a 37 º C.
  6. Coloque dos de 1 cm de vidrio pre-filtro (Whatman, 1823010) en una columna de Costar Spinx (Corning Incorporated, 8.161). La transferencia de la porción líquida de la muestra a la columna. Giro de 1 min a 13.000 rpm en un tubo de 1.5 ml.
  7. Añadir 0,5 l glycoblue y 40 l de acetato de sodio pH 5,5, a continuación, mezclar la muestra. Añadir 1 ml de etanol al 100%, mezcla de nuevo y se precipitan por la noche a -20 ° C.

12. La ligadura de imprimación para el extremo 5 'del cDNA

  1. Decantar y lavar las muestras (véase 10.1), a continuación, volver a suspender las pastillas en ocho mezcla de ligación l (6,5 l de agua, 0,8 l de 10x CircLigase Buffer II, 0,4 l 50 mM MnCl2 de 0,3 l; Circligase II [Epicentro]) e incubar durante 1 hora a 60 ° C.
  2. Añadir 30 l de mezcla oligo recocido (26 l de agua, 3 Buffer FastDigest l [Fermentas], 1 l cut_oligo [10 m]). Incubar durante 1 min a 95 ° C. Luego disminuir la temperatura cada 20 segundos en 1 ° C hasta 25 ° C se alcanzan.
  3. Añadir 2 l BamHI (Fast Fermentas) e incubar durante 30 min a 37 ° C.
  4. Añadir 50 l de TE y 0,5 l glycoblue y mezclar. Añadir 10 l de acetato de sodio pH 5,5 y mezclar, luego añadir 250 l de etanol al 100%. Mezclar de nuevo y se precipitan por la noche a -20 ° C.

13. Amplificación por PCR

  1. Decantar y lavar las muestras (véase 10.1), a continuación, volver a suspender el pellet en 19 l de agua.
  2. Prepare la mezcla de PCR (19 l cDNA, 1 l de mezcla manual P5/P3 Solexa, 10 M cada uno, 20 Accuprime l Supermix una enzima [Invitrogen]).
  3. Ejecute el siguiente programa de PCR: 94 ° C durante 2 minutos, [94 ° C durante 15 segundos, 65 ° C durante 30 segundos, 68 ° C durante 30 segundos] 25-35 ciclos, 68 ° C durante 3 min, 4 ° C para siempre.
  4. Mix 8 l producto de PCR con 2 l de tampón 5x carga TBE y la carga en un 6% prefabricado gel de TBE (InvitRogen). Tinción del gel con Sybrgreen I (Invitrogen) y analizar con una cámara de gel.
  5. El código de barras en las cartillas Rclip permiten a las muestras de multiplex diferentes antes de la presentación para la secuenciación de alto rendimiento. Presentar 15 l de la biblioteca para la secuenciación y guardar el resto.

14. Enlazador y el primer secuencias

ADN linker Pre-adenilado 3 ':

[Se para el adaptador de ADN de IDT y luego hacer alícuotas de 20μM.]

ADN

15. Los resultados representativos:

Antes de la secuenciación de la biblioteca iClip, el éxito de la experiencia puede ser controlado en dos etapas: la autorradiografía del complejo proteína-ARN después de la transferencia de membrana (paso 8.5) y la imagen de gel de los productos de la PCR (paso 13,4). En la autorradiografía de las muestras de baja RNasa, la radioactividad difusas debe ser visto por encima del peso molecular de la proteína (Figura 2, muestra 4). De alta RNasa muestras, la radiactividad se concentra cerca del peso molecular de la proteína (Figura 2, muestra 3). Cuando no hay anticuerpos se utiliza en la inmunoprecipitación, no hay ninguna señal debe ser detectado (Figura 2, las muestras 1 y 2). Aún más los controles importantes para la especificidad de la inmunoprecipitación o bien omitir la radiación UV o células uso que no expresan la proteína de interés 14.

La imagen de gel de los productos de PCR (paso 13,4) debería mostrar un rango de tamaño que corresponde a la fracción de cDNA (alta, media o baja) purificada en el paso 11.4 (Figura 4, carriles 4-6). Tenga en cuenta que los cebadores de PCR P3Solexa y P5Solexa introducir un adicional de 76 nt con el tamaño de la DNA. Si no hay anticuerpos se utiliza durante la inmunoprecipitación, no correspondientes productos PCR deben ser detectados (Figura 4, carriles 1-3). Primer producto dímero puede aparecer a unos 140 nt.

Para obtener resultados representativos de alto rendimiento de secuenciación y posterior análisis bioinformáticos ver 14.

Figura 1
Figura 1. Representación esquemática del protocolo de iClip. ARN en proteínas son complejos de enlaces cruzados covalentes en vivo, utilizando la radiación ultravioleta (paso 1). La proteína de interés se purifica, junto con el ARN unido (pasos 2-5). Para tener en cuenta la secuencia específica de cebado de la transcripción inversa, un adaptador de ARN se liga a los 3 'del ARN, mientras que el extremo 5' es marcado radiactivamente (pasos 6 y 7). Reticulado ARN-proteína complejos se purifican a partir de ARN libre uso de SDS-PAGE y la transferencia de la membrana (paso 8). El ARN se recupera de la membrana por la digestión de la proteína con proteinasa K dejando un polipéptido que permanecen en la cruz-link nucleótidos (paso 9). La transcripción inversa (RT) trunca en el polipéptido restantes y se introducen dos regiones adaptador escindibles y las secuencias de código de barras (paso 10). Selección del tamaño elimina sin imprimación RT antes de circularización. La linealización siguiente genera plantillas adecuadas para la amplificación por PCR (pasos 11-15). Por último, la secuenciación de alto rendimiento genera lee en el que las secuencias de códigos de barras son inmediatamente seguido por el último nucleótido del cDNA (paso 16). Desde esta posición coloca un nucleótido aguas arriba de la secuencia de nucleótidos reticulado, el sitio de unión se puede deducir con alta resolución.

Figura 2
Figura 2. Autorradiografía de reticulado hnRNP C-ARN complejos mediante electroforesis en gel de desnaturalización y transferencia de la membrana. hnRNP C-ARN complejos se inmuno-purificada a partir de extractos de células utilizando un anticuerpo contra hnRNP C (α hnRNP C, las muestras 3 y 4). ARN fue parcialmente digerido con baja (+) o alto (+ +) la concentración de RNasa. Complejos de cambio hacia arriba desde el tamaño de la proteína (40 kDa) se puede observar (muestra 4). El cambio es menos pronunciada cuando las altas concentraciones de RNasa se utilizaron (muestra 3). La señal radioactiva desaparece cuando no hay anticuerpos se utilizó en la inmunoprecipitación (muestras 1 y 2).

Figura 3
Figura 3. Esquema 6% TBE-urea gel (Invitrogen) para guiar la extirpación de los productos iClip cDNA. El gel se ejecuta durante 40 minutos a 180 V que lleva a un patrón de migración reproducible de ADNc y colorantes (luz y de color azul oscuro) en el gel. Use una hoja de afeitar para cortar (línea roja) de la altura (H), media (M) y bajo (L) las fracciones de cDNA. Comience por cortar en la mitad de la tinta de color azul claro y justo encima de la marca en la cinta de gel de plástico. Divide las fracciones de media y baja y el ajuste de la fracción de alto alrededor de 1 cm por encima del tinte de color azul claro. Use cortes verticales guiados por los bolsillos y el tinte para separar las distintas filas (en este ejemplo 1-4). El carril de la marca (m) puede ser teñido y fotografiado para controlartamaños después del corte. Tamaños de los fragmentos se indican a la derecha.

Figura 4
Figura 4. Análisis de la PCR-amplificación de las bibliotecas iClip cDNA mediante electroforesis en gel. ARN se recuperó de la membrana (Figura 1) se transcriben de forma inversa y el tamaño purificado mediante electroforesis en gel desnaturalizante (Figura 2). Tres fracciones de tamaño de cDNA (alta [H]: 120-200 nt, mediano [M]: 85-120 nt y bajo [L]: 70-85 nt) fueron recuperados, circularizado, re-lineal y de amplificación de PCR. Los productos de PCR de la distribución de diferentes tamaños se puede observar como resultado de los diferentes tamaños de las fracciones de entrada. Desde el primer PCR presenta 76 nt con el cDNA, tamaño debe oscilar entre 196 a 276 nt de alto, 161 a 196 nt de mediano y 146-161 nt de fracciones de tamaño bajo. Los productos de PCR están ausentes cuando no de anticuerpos se utilizó para la inmunoprecipitación (carriles 1-3).

Discussion

Dado que el protocolo iClip contiene una amplia gama de reacciones enzimáticas y purificación, que no siempre es fácil identificar un problema cuando un experimento falla. Para el control de la especificidad de identificar ARN entrecruzar los sitios, uno o más controles negativos se debe mantener durante todo el experimento completo y posterior análisis computacional. Estos controles pueden ser la muestra de no-anticuerpos, las células no-reticulado, o inmunoprecipitación de células knock-out o tejido. Idealmente, estos experimentos de control no deben purificar los complejos ARN-proteína, y por lo tanto no deben dar la señal en el gel SDS-PAGE, y ningún producto detectable después de la amplificación por PCR. De alto rendimiento de secuenciación de las bibliotecas de control debe volver secuencias únicas muy pocos. Desmontables células no se recomiendan como un control de secuencia, ya que las secuencias resultantes todavía corresponden a los sitios de enlace transversal de la misma proteína, que se purifica a partir de células caída en cantidades más pequeñas.

Precauciones deben ser tomadas para evitar la contaminación con productos PCR de los experimentos anteriores. La mejor manera de minimizar este problema consiste en separar espacialmente las medidas pre-y post-PCR. Idealmente, el análisis de los productos de PCR y todos los pasos posteriores se deben realizar en una habitación separada. Además, cada miembro del laboratorio debe utilizar su propio conjunto de buffers y otros reactivos. De esta manera, las fuentes de contaminación puede ser más fácil identificarse.

Disclosures

No hay conflictos de interés declarado.

Acknowledgements

Los autores agradecen a todos los miembros de los laboratorios de Ule, Luscombe y Zupan para la discusión y la asistencia experimental. Damos las gracias a James Hadfield y Matthews Nik de secuenciación de alto rendimiento. Nos gustaría señalar que el método descrito aquí iClip acciones de varios pasos con el protocolo clip original, desarrollado por Kirk Jensen y JU en el laboratorio de Robert Darnell. Este trabajo fue apoyado por la beca europea de investigación del Consejo de 206.726-CLIP para JU y largo plazo Ciencia Human Frontiers programa de becas para JK

Materials

Name Company Catalog Number Comments
For gel electrophoresis and membrane transfer we recommend t he use of XCell SureLock® Mini-Cell and XCell IIâ Blot Module Kit CE Mark (Invitrogen, EI0002), which is compatible with the use of the different precast minigels that are specified throughout the protocol. The brand and order number of all materials used is mentioned during the protocol. The list of enzymes used in the protocol is shown in the table below.
Protein A Dynabeads Invitrogen 10001D use protein G for mouse or goat antibody
RNase I Ambion AM2295 activity can change from batch to batch
T4 RNA ligase I New England Biolabs M0204S
PNK New England Biolabs M0201S
proteinase K Roche Group 03115828001
Superscript III reverse transcriptase Invitrogen 18080044
Circligase II Epicentre Biotechnologies CL9021K
FastDigest® BamHI Fermentas FD0054
AccuPrime™ SuperMix I Invitrogen 12342010 this PCR mix gives the best results in our hands

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  8. Ule, J., Jensen, K., Mele, A., Darnell, R. B. CLIP: A method for identifying protein-RNA interaction sites in living cells. Methods. 37, 376-386 (2005).
  9. Licatalosi, D. D. HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature. 456, 464-469 (2008).
  10. Yeo, G. W. An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Nat Struct Mol Biol. 16, 130-137 (2009).
  11. Urlaub, H., Hartmuth, K., Lührmann, R. A two-tracked approach to analyze RNA-protein crosslinking sites in native, nonlabeled small nuclear ribonucleoprotein particles. Methods. 26, 170-181 (2002).
  12. König, J. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol. 17, 909-915 (2010).

Erratum

Formal Correction: Erratum: iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution
Posted by JoVE Editors on 07/14/2011. Citeable Link.

A correction was made to iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution. There was an error in part 2 of step 3. One of the characters had the incorrect symbol and was corrected to:

"...as well as 2 μl Turbo DNase..."

instead of:

"...as well as 2 ml Turbo DNase..."

Comments

71 Comments

  1. Hi,

    First I would like to say this latest method is really neat. I also like Julian's comment at the end of the video when he said with a big smirk, "You have to perform each of the 64 steps with 100% accuracy". :D That is epic.

    On a more serious note, I am just wondering if anyone can suggest what sort of primer I should use if I want to start by cloning my insert into TOPO vector instead of doing nextGen sequencing. Any help is appreciated.

    Paul

    Reply
    Posted by: Anonymous
    June 9, 2011 - 3:47 AM
  2. Hi Paul, thanks for your fun comment! TOPO cloning dŒsn²17;t require any specific primer, so you could use the one described in the protocol. Unless you wish to do something specific, such as concatemerization of sequences before inserting them into vector. Feel free to post more questions! Jernej

    Reply
    Posted by: Anonymous
    June 11, 2011 - 4:19 PM
  3. For more iCLIP questions and answers, use the following Googledoc: http://goo.gl/4tSci.

    Reply
    Posted by: Anonymous
    June 13, 2011 - 11:28 AM
  4. Hi Jernej,
    Is it possible to use a 3' linker with a phosphorylated 5' end instead of a pre-adenylated 5' end and adding some ATP during the 3' linker ligation step? Thanks. Paul

    Reply
    Posted by: Anonymous
    June 13, 2011 - 10:13 PM
  5. Yes, just follow the protocol as described in Konig et al, NSMB ²010 (PMID ²0601959). More on Googledoc.

    Reply
    Posted by: Anonymous
    June 14, 2011 - 3:48 AM
  6. Hi Jernej,

    Sorry to keep bombarding you with questions. In the supplementary section of your NSMB ²010 paper, shrimp alkaline phosphatase was used to desphosphorylate 3' ends. My understanding is that SAP can only desphosphorylate 5' ends. I am wondering if you had dephosphorylated 3' ends step using PNK before using SAP to dephosphorylate 5' ends.

    Quote from Konig et al, NSMB ²010: "For dephosphorylation of 3²4²; ends, Dynabeads were resuspended in ² µl 10&#²15; Shrimp alkaline phosphatase buffer (Promega), 17.5 µl H²O and 0.1 µl Shrimp alkaline
    phosphatase (Promega) and incubated at 37°C for 10 min with intermittent shaking (10 sec at 700 rpm followed by ²0 sec pause)."

    Thank you again for your help.

    Reply
    Posted by: Anonymous
    October 5, 2011 - 4:04 AM
  7. We did use SAP in the NSMB protocol - it dŒsn't work as well as PNK on the 3' ends. We couldn't use PNK at the time, because PNK carryover into ligation reaction would create problems in the presence of ATP. In jove protocol, ligation reaction lacks ATP, therefore we can use PNK to dephosphorylate the 3' ends.

    Reply
    Posted by: Anonymous
    October 5, 2011 - 6:30 PM
  8. Hi Jernej,

    On 3.² it says add ²ml Turbo DNAse into the 1.5 ml tube. I am wondering if that amount is correct.

    Reply
    Posted by: Anonymous
    July 9, 2011 - 11:45 PM
  9. Hello Paul,
    you are right, it should be two micro liters. Sorry for that, I will try to have it changed,
    Julian

    Reply
    Posted by: Anonymous
    July 10, 2011 - 6:56 AM
  10. Hi Jernej, great protocol! Just a precision, the L3 oligo is a pre-adenylated DNA or RNA oligo? Not clear as the original Clip and iClip uses RNA...

    Thanks a bunch,

    Marco

    Reply
    Posted by: Anonymous
    September 12, 2011 - 3:58 PM
  11. Hi Marco. It's a DNA oligo. Best, Jernej

    Reply
    Posted by: Anonymous
    September 12, 2011 - 4:02 PM
  12. Hi Jernej,

    I am wondering if the 3²P-ATP batch that you normally use in your lab for step 6.1 always has close to a 100% reported radioactivity. What is the lowest percentage of remaining 3²P that you can usually still get away with? I can still get some decent signal when using 3²P-ATP that has ~50-60% remaining radioactivity but my bands on the films are not as intense as the one that I see in your publications. I am trying to work out the best schedule for ordering some 3²P-ATP and starting my experiments. Thanks again.

    Paul

    Reply
    Posted by: Anonymous
    September 14, 2011 - 11:32 PM
  13. We don't use ATP if it's more than two weeks old, thus we have >50% radioactivity. But signal intensity also depends on the efficiency of crosslinking and IP,and amount of protein expression in the cells.

    Reply
    Posted by: Anonymous
    September 15, 2011 - 4:23 AM
  14. Hi Jernej,
    Thank you for the protocol. What results if I reduce the cell samples to 100-1000 (not 10*6-7 cells) ? Thanks for your reply.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 12:24 AM
  15. That would be challenging. If you have an abundant protein that cross-links well to RNA, then it might be possible. So try running the radioactive protein-RNA complex on the gel - if you good signal after overnight exposure, then it's doable.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 4:52 AM
  16. Hi, Jernej !
    Thank you for the reply. I have another questions: How stable if the RNA-RNA and RNA-Protein photocrosslinking? How to degrade these proteins or remove the photocrosslinking? Thank you a lots.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 6:36 AM
  17. Hi Jernej,

    I again have some more questions. Do you still expose the nitrocellulose membrane at -80C when using phosphoimager instead of a film? I'm also wondering what exposure time your lab uses when using a phosphorimager screen.

    Secondly, I am wondering how many libraries containing different barcodes you can run together in a single flow cells.

    Thank you again Jernej. This protocol has been extremely useful.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:49 PM
  18. Cross-linking forms a covalent bond, so is irreversible (read the paper!). -80 would ruin the phosphorimager screen, so don't do it! We normally multiplex ±10 libraries.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:53 PM
  19. Cross-linking forms a covalent bond, so is irreversible (read the paper!). -80 would ruin the phosphorimager screen, so don't do it! We normally multiplex ±10 libraries.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:53 PM
  20. Cross-linking forms a covalent bond, so is irreversible (read the paper!). -80 would ruin the phosphorimager screen, so don't do it! We normally multiplex ±10 libraries.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:53 PM
  21. Hi Jernej,

    In regards to one of the FAQs from Google docs.

    - When analysing PCR products, I see a band corresponding to the size of primer dimers, especially in the sample that was cut low from cDNA gel.

    Yes, it is common to see this band in the sample that was cut low from cDNA gel, and sometimes also in other samples. This is due to contamination from short cDNAs that only contain the sequence of RT primer. If this primer dimer is the dominant product on gel, we advise against sequencing the corresponding sample.

    I seem to be getting this short cDNA contamination all the time. Do you have any advice on how I could try to minimise the contamination? Have you ever isolated fragments of correct-size cDNA from a TBE-urea gel and sent only the isolated fragment for sequencing when you have short cDNA contaminations? Do you think that will work? I think that the concentration of L3 linker that I had used might have been too much. Thank you.

    Reply
    Posted by: Anonymous
    November 22, 2011 - 7:45 PM
  22. There are several possible reasons for this. Maybe one aspect of the protocol is not working, and therefore you are not producing any specific cDNA. If you have no cDNA input, then with enough cycles, you can amplify the primer-primer from any part of the gel. If you are using mammalian cells, try to get the protocol working first with hnRNP C or TIA with Santa cruz antibodies that we used in recent publications. Otherwise, using too much L3 can be a problem.

    Reply
    Posted by: Anonymous
    November 23, 2011 - 4:35 AM
  23. Very useful protocol. I have two questions.

    1. For dephosphorylation of RNA 3'ends, pH 6.5 PNK buffer is used, rather than the pH 7.6 buffer, provided by NEB. Have you compared these two conditions internally?
    ². In the protocol, the final PCR product is not isolated and quantitated before submitting for the sequencing. Are there any potential problems of doing these two steps? Can I isolate the PCR product and re-PCR using the same primers to get more product (for Illumina Hiseq)? Thank you.

    Reply
    Posted by: Anonymous
    January 5, 2012 - 3:40 PM
  24. You can find more related answers in Googledoc http://goo.gl/4tSci, but short answers are also below:

    1. We haven²17;t compared conditions, but increased phophatase activity of PNK at lower pH has been reported in literature, you can read more in the Pubmed ID 1184²1²0.

    ². The PCR product needs to be quantified. We use both qPCR and bioanalyser. Normally, the products of the first PCR should look clean on the gel, otherwise it is a sign of a library that is of low complexity, and is unlikely to generate informative data. Therefore we advise against re-PCR, but it can be done as the last resource.

    Reply
    Posted by: Anonymous
    January 5, 2012 - 4:56 PM
  25. Hi Jernej,

    I noticed you use +/- 10 multiplexed libraries; I was wondering if you knew how many are necessary for a successful run (i.e. to provide sufficient distribution for cluster identification)?

    Reply
    Posted by: Anonymous
    March 29, 2012 - 2:20 PM
  26. The way the primers are designed here, no multiplexing is necessary, because the first three nucleotides in the primer sequence are random (part of randomer = NNN).

    Reply
    Posted by: Anonymous
    March 29, 2012 - 2:28 PM
  27. I appreciate your experiment. I have some qeustions.

    In this protocol, what dŒs barcode do high-throughout squencing?

    I don't understand function of barcode



    Reply
    Posted by: seung kuk P.
    May 23, 2012 - 6:45 AM
  28. Hi,
    this might be a really naive question but I'm wondering at the UV cross linking step, when you say you irradiate once, dŒ's this mean 1 min?

    Thank you!
    Zsofi

    Reply
    Posted by: Zsofia I.
    June 18, 2012 - 1:19 PM
  29. Hello, Thank you for this helpful technique, I just have a question. My experiments protocols are: 1. UV-crosslink RNA-protein; ². Isolate the RNA-protein complex by immunopricitation; 3. Isolate the binding RNA. 3²P-labeling the binding RNA. 4. Analysis the RNA by microarray.
    Because I do not need to sequence the RNA, and I only want to isolate the binding RNA for microarray analysis after UV-crosslink RNA-protein, so I wonder whether I need to do the step 5-7 in your protocols or I could skip from step 4 to step 8 in your protocol?

    Thanks very much, I look forward to your kind reply!

    Sean

    Reply
    Posted by: xiaoyun w.
    July 22, 2012 - 9:09 PM
  30. It is unlikely you will have enough cDNA for microarray hybridisation without some kind of amplification. You can try using steps 4-8, but you could also amplify in other ways.

    Reply
    Posted by: Anonymous
    July 23, 2012 - 6:03 AM
  31. Hi Jernej,

    Is there any published article on how to analyse iCLIP's high-throughput sequencing data? I have just got my sequencing results back following steps in your protocol. I want to make sure I check with you before digging into the data. Thank you.

    Reply
    Posted by: Anonymous
    August 1, 2012 - 10:15 PM
  32. The article is not yet published, but is in preparation by Tomaz Curk ( http://www.fri.uni-lj.si/en/tomaz-curk/), who made a public server: http://icount.biolab.si/. You can contact Tomaz at tomaz.curk@fri.uni-lj.si for more information.

    Reply
    Posted by: Anonymous
    August 2, 2012 - 5:47 AM
  33. Hi,
    it is so powerful technique! But I cannot IP any protein follow protocol. Is there any difference in affinity between different antibodies and their antigen? Could you give me some advice? Maybe we could decrease concentration of SDS or sodium deoxycholate?
    Thanks, I look forward to your kind reply!
    Min

    Reply
    Posted by: Min S.
    August 5, 2012 - 11:04 PM
  34. Hi Min, you can find advice on IP googledoc http://goo.gl/4tSci.

    Reply
    Posted by: Anonymous
    August 6, 2012 - 3:35 AM
  35. Hi Jernej,

    With the barcoding system, I am just wondering if the three random nucleotides are there for indexing purpose during Illumina sequencing run but it's not necessary for splitting the different libraries later on. For RC1, the sequencing results will be something like NNNGGTTNN.... During analysis, do you usually trim the 3-bp from the 5'-end of the results and split the different replicates after the trimming step? I have just realised this was slightly different to the barcoding system used in your NSMB paper. -paul

    Reply
    Posted by: Anonymous
    August 6, 2012 - 9:41 PM
  36. Hi Paul! You can find the answer under the topic of "Use of random barcode in data analysis" in http://goo.gl/4tSci.

    Reply
    Posted by: Anonymous
    August 7, 2012 - 7:58 AM
  37. Hi Jernej,

    I started optimising CLIP couple of months ago and I'm at the stage that I'm convinced that I can efficiently cross link RNA to my protein (checked it by specific qRT PCR). I'm lucky because I don't need to fiddle with the IP since I've optimised before and works fine. But just to double check, after IP and western blotting a smear and a lower amount of original kDa protein is a good sing for cross linking yes?
    So my problems started at the RNase A step, I don't see any changes in size/appearance on WB after treatment... I'm convinced that my protein creates a massive complex (couple of 100 kDa) and it is because my target RNA is 10 kb to start with and there are at least 3 proteins binding to it. I'm working with a RNA virus, that's the explanation for it. I think the reason I don't see any change in kDA is because the complex dŒsn't even enter the gel to start up with. Although I used the given buffer which should break any membrane apart but the proteins are still there possibly protecting the RNA. Did you ever come across similar problems and would you have any suggestions? Also, I understand that the RNase trimming is necessary for the efficient RT step but is it a problem if the RNA is too long? What is too long? DŒs this depend on the RT enzyme used I recon or is this also important for the sequencing?

    I would greatly appreciate yur help because I'm stuck...

    Thank you,
    Zsofi

    Reply
    Posted by: Zsofia I.
    October 10, 2012 - 7:38 AM
  38. Hi Zsofi,

    For partial RNAse digestion we use RNase I (step 3). We use two different concentrations: a lower one that makes fragments with a mean between 50-100 bp and a higher concentration that fragments RNA to around 10bp. The lower one is used for preparing libraries, the higher one is used for analytical reasons.

    The RNAse step is important to (1) allow the protein RNA complex enter the Gel (²) to narrow down the crosslink site to a fragment with a size compatible with high throughput sequencing (maximum around 300 bp). So you definitely need to optimize this step for your experiments.

    If the complex you are studying is not covalently linked it should fall apart during the denaturing Gel run. Only a small fraction of your complex will have all the proteins of your complex crosslinked to the RNA at the same time since crosslinking is a very inefficient step. Therefore with the higher RNAse concentration you should be able to see a radioactive signal at the size of the protein you are studying.

    I hope that helps, best regards,
    Julian

    Reply
    Posted by: Julian K.
    October 11, 2012 - 9:49 AM
  39. Hi Julian,

    I have had some trouble with the RNase step when nuclease-ing the total lysate... In my troubleshooting efforts I read that RNase I is inhibited by 0.1% SDS, which is the concentration used in your lysis buffer. It dŒsn't seem that you guys have any problem though...do you think this is due to using an excess of RNase I or what? Just curiously confused. Thanks,

    sam

    Reply
    Posted by: Sam F.
    February 5, 2013 - 6:00 PM
  40. Hi Sam,

    in our experience the inhibition of RNase I by SDS is not an issue. You just optimize the concentration of RNase I to obtain the desired fragmentation. If you have problems doing that with your buffer conditions, you could also do the RNase digestion on the beads instead of in the lysate.

    Best,
    Julian

    Reply
    Posted by: Julian K.
    February 6, 2013 - 6:19 AM
  41. Thanks for the quick reply Julian. Your recommendation to do the "on bead" digestion is exactly what I have done and it seems to be working fine. Cheers

    Posted by: Sam F.
    February 6, 2013 - 10:12 AM
  42. Hi,

    I was wondering how many minutes have you irradiated the cells in case of HNRNP C?

    Reply
    Posted by: Niaz M.
    November 26, 2012 - 6:29 PM
  43. Hi Niaz,
    we are normally not measuring time of irradiation but the Energy per square centimeter:
    Step 1.²: ... Irradiate once with 150 mJ/cm² at ²54 nm.
    In our Stratalinker this takes 50s. However time of irradiation is not very informative here since it changes with the age or quality of the lamps, etc.
    Cheers, Julian

    Reply
    Posted by: Julian K.
    November 27, 2012 - 6:20 AM
  44. Hi,

    Thank you for wonderful protocol !

    I would like to confirm about adaptor and primer sequences.
    1. L3 adaptor and Rclip RT primer has ²²0;same²²1; sequences, not ²²0;complementary²²1; sequences. Are they O.K.? In my understanding, L3 and TR primers should have ²²0;complementary sequences.
    ². P3 Solexa 3²17; 11 nt sequence (TCTTCCGATCT) looks ²²0;extra²²1;. Both of P5 and P3 have the same sequence, which is complementary to Rclip RT primer or L3 adaptor. I think only P5 should have this sequence.

    Thank you for your help.

    Best,
    Lisa

    Reply
    Posted by: Risa K.
    December 18, 2012 - 3:04 AM
  45. Hi,

    Thank you for wonderful protocol !

    I would like to confirm about adaptor and primer sequences.
    1. L3 adaptor and Rclip RT primer has ²²0;same²²1; sequences, not ²²0;complementary²²1; sequences. Are they O.K.? In my understanding, L3 and TR primers should have ²²0;complementary sequences.
    ². P3 Solexa 3²17; 11 nt sequence (TCTTCCGATCT) looks ²²0;extra²²1;. Both of P5 and P3 have the same sequence, which is complementary to Rclip RT primer or L3 adaptor. I think only P5 should have this sequence.

    Thank you for your help.

    Best,
    Lisa

    Reply
    Posted by: Risa K.
    December 18, 2012 - 3:04 AM
  46. Hi Lisa,

    it is correct that the ends of P3 and P5 primers are the same. This is because of Illumina's primer design for their high throughput sequencing platform. When you look at the 3' end of the Rclip primers (after the Bamhi cleavage site) you can see that they are actually complementary to the 3'end of the L3 adapter.

    Cheers,
    Julian

    Reply
    Posted by: Julian K.
    December 18, 2012 - 10:05 AM
  47. I got it !!!
    Thank you :)

    Best,
    Lisa

    Reply
    Posted by: Risa K.
    December 18, 2012 - 1:11 PM
  48. Hi
    Thanks for the protocol. I have one question that has been bothering me, though. Both the RNA ligase and PNK buffers will expose the antibody column to relatively high dithiothreitol (DTT) concentrations (10 mM and 5 mM respectively). Why dŒsn't this destroy the column by reducing the disulphide bonds holding the heavy and light antibody chains together? Have you ever tried to improve the immunoprecipitation step by attempting to minimize the DTT concentration as much as possible or is this not an issue. Any assistance would be greatly appreciated. Thanks - Greg

    Reply
    Posted by: Greg C.
    February 3, 2013 - 2:13 PM
  49. Hi Greg, we haven't seen an effect of the DTT in the buffers on the IP efficiency, it seems that the concentration is not high enough to reduce the IgG - however, it is worth testing this the first time you do IP, since it is plausible that this will vary dependent on the source of your buffers (company used for PNK and ligase), or antibodies.

    Reply
    Posted by: Anonymous
    February 6, 2013 - 2:45 AM
  50. Hi Jernej,
    Thanks for the reply. The antibody I am using is definitely sensitive to the level of DTT found in the PNK buffer and I need to limit the over-all exposure of the column to DTT as much as possible. As a result, rather than using PNK as the 3' phosphatase, I would like to use an alkaline phosphatase. I noticed that in your ²010 NSMB paper you are using Shrimp Alkaline Phosphatase and in your ²009 Methods paper you use FAST AP. Did you find that the Shrimp phosphatase is significantly better ?

    Thanks again - Greg

    Reply
    Posted by: Greg C.
    February 8, 2013 - 3:52 PM
  51. Hi Greg, we don't have any evidence to suggest that one is better than the other for the on-bead reaction. At the time we were using SAP in the lab generally since it can be heat-inactivated, so therefore we also used it for on-bead (even though here you can't heat-inactivate it on beads). So you can go ahead with either one.

    Reply
    Posted by: Anonymous
    February 9, 2013 - 5:29 AM
  52. I should also add that even though we didn't compare FAST AP and SAP, we did compare SAP with PNK, and we had a lot better results with PNK. It seems that SAP is not efficient as a 3' phosphatase. So it may be better for you to determine the minimal DTT amount in the buffer that is compatible with your antibody, and then continue using it with PNK and ligase. If you use fresh DTT, 1mM is likely to be sufficient both for PNK and RNA ligase.

    Reply
    Posted by: Anonymous
    February 9, 2013 - 5:39 AM
  53. Thanks, I really appreciate the advice.
    -Greg

    Posted by: Greg C.
    February 9, 2013 - 9:03 AM
  54. Hi, thanks for the awesome video. I have two questions related to the reagents:
    1. What concentration is the PEG400? (it only says 4 ul in the protocol).
    ². Under "Reverse transcription", step 6, what is the pH of the TE buffer you use? Is it pH 8?
    Thank you very much for your help. -QT

    Reply
    Posted by: Qiumin T.
    February 7, 2013 - 1:22 PM
  55. Hi QT,
    (1) we are using PEG400 from Sigma (²0²398). It is a viscous liquid.
    (²) Yes, the it is pH 8
    Cheers, Julian

    Reply
    Posted by: Julian K.
    February 7, 2013 - 4:56 PM
  56. Thank you so much Julian. I have another question. Could you recommend a protocol for doing iCLIP with mouse brain tissue? Do you know whether the tissue prep steps from this protocol ( http://ago.rockefeller.edu/Ago_HITS_CLIP_Protocol_June_²009.pdf) will work well for iCLIP as well?

    Reply
    Posted by: Anonymous
    February 13, 2013 - 5:52 PM
  57. This protocol should be fine. We also recently published a bookchapter about the iCLIP protocol which contains information on tissue samples and lots of other useful info and background:
    http://onlinelibrary.wiley.com/doi/10.100²/97835²764458².ch10/summary

    Reply
    Posted by: Julian K.
    February 14, 2013 - 5:19 AM
  58. The pre-publication version of the book chapter is available here: http://www².mrc-lmb.cam.ac.uk/groups/jule/publications/Konig_wiley.pdf.

    Reply
    Posted by: Anonymous
    February 19, 2013 - 1:54 PM
  59. I enjoyed reading about your updated iCLIP protocol in the book Tag-based Next Generation Sequencing from Wiley. I would be grateful for more details about the amount and activity of the 3²-P that you use to radioalabel the RNA. In both the book chapter and the JoVE article I see only volumes, not activities.

    In the figure for step 9, the radiolabel on the 5' end of the RNA is missing, but shouldn't it still be there? At what point in the protocol can we be reasonably sure that we are dealing with unlabeled material?

    Also, have you ever explored non-radioactive approaches to labeling, or is the sensitivity of these methods too low for the purposes of this protocol?

    Thanks!

    John

    Reply
    Posted by: John S.
    June 17, 2013 - 9:23 AM
  60. Dear John,

    with the current protocol most of the radioactivity is gone after the gel purification of the cDNA. You can increase this effect by treating the samples with RNAse after the reverse transcription (The radioactive RNA fragments then running much faster then the cDNAs in the gel). We are currently working on a protocol where we fragment the RNA by alkaline hydrolysis, which will be available soon (We want to avoid using too much RNAse at our desks).

    In addition you should always measure your samples with a Geiger counter. If your final PCRs are still hot, then you should decrease the fraction of beads that go into the labeling reaction.

    Best wishes,
    Julian

    Reply
    Posted by: Julian K.
    June 19, 2013 - 11:52 AM
  61. Hi,
    I have a question which relies on your experience with the data generated with CLIP:
    because of the UV irradiation the protein crosslinks to the RNA which even after proteinase K treatment presents an obstruction to the reverse transcriptase which therefore either skips or adds random nucleotide(s). So my question is that how long the deletions/insertions can be? Is it only one nucleotide or can also be 20?

    Thank you very much!!!

    Reply
    Posted by: Zsofia I.
    August 8, 2013 - 12:06 PM
  62. We see that >80% of cDNAs truncate at the crosslink site, and the mutations are quite rare in the remaining sequences. All we know about crosslink-induced mutations has been published here: http://www.ncbi.nlm.nih.gov/pubmed/22863408.

    Reply
    Posted by: Anonymous
    August 8, 2013 - 12:15 PM
  63. Thanks for your protocol. I have a question about the IgG background signal in the p32 labeled Western Blot. I used mouse IgG1 isotype as a control. I did not crosslink the IgG to the beads, so IgG1 stays around 50 and 25 KDa region. I do observe some radioactivity band around 50 kDa. Do you notice this in your experiments as well? Since it is close to my protein region, can you give me some suggestions to avoid this?

    Sincerely,
    Mei

    Reply
    Posted by: Xuemei Z.
    August 9, 2013 - 1:42 PM
  64. We don't get a signal in control IP. Most likely this is an RBP that non-specifically binds under your conditions. It is important to wash with high-salt buffer, and rotate the tubes for ±5min during these washes. Also, diluting the lysate before IP may help. Standard IP optimisations, basically.

    Reply
    Posted by: Anonymous
    August 9, 2013 - 2:24 PM
  65. Hi,
    Thank you for wonderful protocol.
    Usually how much RNA concentration one should get after Isolation from membrane? I would appreciate your reply.

    Reply
    Posted by: Bhagya B.
    August 13, 2013 - 5:29 AM
  66. Hi,
    Thank you for wonderful protocol.
    Usually how much RNA concentration one should get after Isolation from membrane? I would appreciate your reply.

    Reply
    Posted by: Bhagya B.
    August 13, 2013 - 5:57 AM
  67. Hi,
    I have 2 more questions. In this protocol you did not remove 5' phosphate of the RNA, can you still label the 5' side with P32 by PNK later? Another question, is it possible to just p32 label the RNA, cut the band, extract, degrade the protein and add 3' linker for RT later?

    Reply
    Posted by: Xuemei Z.
    August 21, 2013 - 2:06 PM
  68. Normal PNK has phosphatase activity, so it can replace the 5' phosphate. The original CLIP protocol from Ule et al, Science 2003 added 3' linker after RNA extraction, but as explained in Ule et al, Methods 2005., the efficiency and purity of the protocol increases if linker is ligated on beads.

    Reply
    Posted by: Anonymous
    August 21, 2013 - 2:15 PM
  69. Thanks for this amazing protocol and your rapid and very helpful exchange here in this site.

    Reply
    Posted by: Xuemei Z.
    August 21, 2013 - 2:26 PM
  70. Hello, thank you for this wonderful protocol.
    I have a question:
    -I get positive Radioactive signal at the right size of positive CTRL used in this protocol in the NOT UV samples, it looks exactly as I was using high RNAse condition. why?
    I am phosphorylating the protein? is it possible?
    thank you

    Reply
    Posted by: jessica c.
    February 27, 2016 - 8:45 PM
  71. Hi Jessica, you are right, if you see signal in the non-UV control, this means that the protein is getting phosphorylated in some other way. If it has a kinase domain it may even phosphorylate itself. Or maybe some kinase is getting co-purified? You could check for this by omitting PNK from the phosphorylation reaction.

    Reply
    Posted by: Jernej U.
    March 16, 2016 - 11:07 AM

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