{"id":25667,"date":"2022-02-13T13:44:18","date_gmt":"2022-02-13T05:44:18","guid":{"rendered":"https:\/\/www.accscicn.com\/?p=25667"},"modified":"2022-10-20T17:57:11","modified_gmt":"2022-10-20T09:57:11","slug":"application-mantis-01","status":"publish","type":"post","link":"https:\/\/www.accscicn.com\/es\/vision\/aplicacion\/aplicacion-mantis-01\/","title":{"rendered":"Simplificaci\u00f3n de la preparaci\u00f3n de bibliotecas NEXTERA XT para la secuenciaci\u00f3n de ARN de muestras de ADNc de c\u00e9lulas neuronales de rat\u00f3n mediante el procesador de l\u00edquidos MANTIS"},"content":{"rendered":"<div data-elementor-type=\"wp-post\" data-elementor-id=\"25667\" class=\"elementor elementor-25667\" data-elementor-post-type=\"post\">\n\t\t\t\t\t\t<section class=\"elementor-section elementor-top-section elementor-element elementor-element-786eb2fc elementor-section-full_width elementor-section-stretched elementor-section-height-default elementor-section-height-default\" data-id=\"786eb2fc\" data-element_type=\"section\" data-settings=\"{&quot;stretch_section&quot;:&quot;section-stretched&quot;,&quot;background_background&quot;:&quot;classic&quot;}\">\n\t\t\t\t\t\t<div class=\"elementor-container elementor-column-gap-default\">\n\t\t\t\t\t<div class=\"elementor-column elementor-col-100 elementor-top-column elementor-element elementor-element-45dc627e\" data-id=\"45dc627e\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap elementor-element-populated\">\n\t\t\t\t\t\t<section class=\"elementor-section elementor-inner-section elementor-element elementor-element-396ffb01 elementor-section-boxed elementor-section-height-default elementor-section-height-default\" data-id=\"396ffb01\" data-element_type=\"section\">\n\t\t\t\t\t\t<div class=\"elementor-container elementor-column-gap-default\">\n\t\t\t\t\t<div class=\"elementor-column elementor-col-50 elementor-inner-column elementor-element elementor-element-1ed8d15\" data-id=\"1ed8d15\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap elementor-element-populated\">\n\t\t\t\t\t\t<div class=\"elementor-element elementor-element-1dd956c elementor-widget elementor-widget-page-title\" data-id=\"1dd956c\" data-element_type=\"widget\" data-widget_type=\"page-title.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t\t\t\n\t\t<div class=\"hfe-page-title hfe-page-title-wrapper elementor-widget-heading\">\n\n\t\t\t\t\t\t\t\t\t\t\t<a href=\"https:\/\/www.accscicn.com\/es\">\n\t\t\t\t\t\t<h3 class=\"elementor-heading-title elementor-size-default\">\n\t\t\t\t\t\t\t\t\n\t\t\t\tSimplificaci\u00f3n de la preparaci\u00f3n de bibliotecas NEXTERA XT para la secuenciaci\u00f3n de ARN de muestras de ADNc de c\u00e9lulas neuronales de rat\u00f3n mediante el procesador de l\u00edquidos MANTIS  \n\t\t\t<\/h3> \n\t\t\t\t\t\t\t\t\t<\/a>\n\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<div class=\"elementor-element elementor-element-cb0ab86 elementor-widget elementor-widget-text-editor\" data-id=\"cb0ab86\" data-element_type=\"widget\" data-widget_type=\"text-editor.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t<style>\/*! elementor - v3.21.0 - 20-05-2024 *\/\n.elementor-widget-text-editor.elementor-drop-cap-view-stacked .elementor-drop-cap{background-color:#69727d;color:#fff}.elementor-widget-text-editor.elementor-drop-cap-view-framed .elementor-drop-cap{color:#69727d;border:3px solid;background-color:transparent}.elementor-widget-text-editor:not(.elementor-drop-cap-view-default) .elementor-drop-cap{margin-top:8px}.elementor-widget-text-editor:not(.elementor-drop-cap-view-default) .elementor-drop-cap-letter{width:1em;height:1em}.elementor-widget-text-editor .elementor-drop-cap{float:left;text-align:center;line-height:1;font-size:50px}.elementor-widget-text-editor .elementor-drop-cap-letter{display:inline-block}<\/style>\t\t\t\t<h4>Presentaci\u00f3n<\/h4><p>Los laboratorios centrales deben equilibrar la eficiencia de sus flujos de trabajo de NGS con la calidad de los datos que generan al proporcionar una variedad de servicios de secuenciaci\u00f3n de pr\u00f3xima generaci\u00f3n (NGS) relacionados con una amplia gama de clientes, tipos de muestras y m\u00e9todos de secuenciaci\u00f3n. A medida que disminuye el coste de la secuenciaci\u00f3n, los costes asociados a la preparaci\u00f3n de bibliotecas est\u00e1n empezando a convertirse en una parte importante de los costes generales del flujo de trabajo de NGS. Para hacer frente a este importante problema de costes en la secuenciaci\u00f3n, muchas instituciones han seguido la tendencia hacia la miniaturizaci\u00f3n de las reacciones, reduciendo de hecho los vol\u00famenes de reacci\u00f3n al volumen recomendado por el fabricante de 10%.<\/p><p>Esta nota de aplicaci\u00f3n destaca el flujo de trabajo para generar bibliotecas NGS de alta calidad mediante la dispensaci\u00f3n precisa de reactivos microflu\u00eddicos de desplazamiento positivo, incluso en presencia de un bajo aporte celular. Los datos de control de calidad de una preparaci\u00f3n de bibliotecas Illumina Nextera XT de un cuarto de volumen demuestran la aplicabilidad y la eficacia de la miniaturizaci\u00f3n de la reacci\u00f3n en el contexto de la secuenciaci\u00f3n unicelular.<\/p><p>Materiales.<\/p><ul><li>Placa Eppendorf twin.tec 384 (PN 0030129342)<\/li><li>Kit de preparaci\u00f3n de bibliotecas de ADN Illumina Nextera XT (PN FC-131-1096)<\/li><li>Kit de indexaci\u00f3n Illumina Nextera XT V2 (FC-131-2001)<\/li><li>Termociclador<\/li><li>Procesador de l\u00edquidos FORMULATRIX\u00ae MANTIS\u00ae (PN MANTV3.2)<\/li><li>Alpaqua 384 Columna Placa Magn\u00e9tica (PN A001222)<\/li><li>Fluor\u00f3metro ThermoFisher Qubit 4 (PN Q33226)<\/li><li>Bioanalizador Agilent 2100 (PN G2939BA)<\/li><\/ul><h4>M\u00e9todos<\/h4><p>Preparaci\u00f3n de bibliotecas Illumina Nextera XT de un cuarto de volumen<\/p><p>En este proyecto piloto se utilizaron ocho muestras de ADNc de c\u00e9lulas neuronales de rat\u00f3n (3 tipos celulares; 1 ng de ADN de entrada), y a continuaci\u00f3n se evalu\u00f3 la calidad de la biblioteca generada en un cuarto de volumen mediante el siguiente protocolo experimental:<\/p><p>**A. Tagment cDNA - volumen total de reacci\u00f3n de 6,35 \u00b5L **<\/p><ol><li><p>A\u00f1ada 1,25 \u00b5l de ADN de entrada a los pocillos de una placa de microtitulaci\u00f3n de 384 pocillos.<\/p><\/li><li><p>El tamp\u00f3n TD se pipetea en una punta de pipeta de 200 \u00b5l* y se coloca en el chip LV del MANTIS. Dispense 2,5 \u00b5l de tamp\u00f3n TD en cada pocillo que contenga una muestra en la microplaca MANTIS.<\/p><\/li><li><p>Mezcla.<\/p><\/li><li><p>Se pipetea ATM en una punta de pipeta de 200 \u00b5L* y se coloca en el chip LV del MANTIS. Dispensar 1,3 \u00b5l de ATM en cada pocillo de la microplaca MANTIS que contiene la muestra.<\/p><\/li><li><p>Mezcla.<\/p><\/li><li><p>Ciclo t\u00e9rmico.<\/p><p>i. 5 minutos a 55\u00b0C<\/p><p>ii. tras lo cual se conserva a 10\u00b0C<\/p><\/li><li><p>El tamp\u00f3n NT se pipetea en una punta de pipeta de 200 \u00b5l* y se coloca en el chip LV del MANTIS. Dispense 1,3 \u00b5l de tamp\u00f3n NT en cada pocillo que contenga una muestra en la microplaca MANTIS.<\/p><\/li><li><p>Mezcla.<\/p><\/li><li><p>Incubar a temperatura ambiente durante 5 minutos.<\/p><\/li><\/ol><p><strong>B. Amplificaci\u00f3n de la biblioteca - 12,65 \u00b5l de volumen total de reacci\u00f3n<\/strong><\/p><ol><li><p>Pipetear el MNP en una punta de pipeta de 200 \u00b5l* y colocarla en el chip LV del MANTIS. Dispense 3,8 \u00b5l en cada pocillo que contenga una muestra en la microplaca MANTIS.<\/p><\/li><li><p>Se pipetearon manualmente 1,25 \u00b5l de ambos cebadores \u00edndice en las muestras para obtener una combinaci\u00f3n \u00fanica de cebadores \u00edndice para cada muestra.<\/p><\/li><li><p>Mezcla.<\/p><\/li><li><p>Ciclo t\u00e9rmico.<\/p><p>i. 3 minutos a 72\u00b0C<\/p><p>ii. 30 segundos a 95\u00b0C<\/p><p>iii. realizar los 12 ciclos siguientes<\/p><p>a. 10 segundos a 95\u00b0C<\/p><p>b. 30 segundos a 55\u00b0C<\/p><p>c. 30 segundos a 72\u00b0C<\/p><p>iv. 5 minutos a 72\u00b0C<\/p><p>v. 10\u00b0C en todo momento<\/p><\/li><\/ol><p><strong>C. Limpieza de la biblioteca<\/strong><\/p><p>1. Extraer las microesferas AMpure XP en una punta de pipeta de 200 \u00b5L* y colocar en el chip HV del MANTIS. 2. Dispensar 10 \u00b5L en cada pocillo de la microplaca MANTIS que contiene la muestra.<\/p><p>2. Mezcla.<\/p><p>3. Incubar a temperatura ambiente durante 5 minutos<\/p><p>4. Colocar la microplaca sobre el im\u00e1n y esperar 2 minutos<\/p><p>5. Eliminar todo el sobrenadante manualmente (no retirar la microplaca del im\u00e1n hasta el paso 11).<\/p><p>6. Utilizando la opci\u00f3n de funci\u00f3n de flujo continuo del MANTIS, a\u00f1ada 50 \u00b5l de 80% EtOH a cada pocillo que contenga la muestra en la microplaca.<\/p><p>7. Incubar durante 30 segundos.<\/p><p>8. Eliminar manualmente todo el sobrenadante mientras se coloca la placa de microtitulaci\u00f3n en el im\u00e1n.<\/p><p>9. Repita los pasos 6-8. 1. Deje secar al aire durante 15 minutos. 2. Retire la microplaca del im\u00e1n. 3. Pipetear el RSB en una punta de pipeta de 200 \u00b5L* y colocar en el chip HV del MANTIS. 4. Dispensar 15 \u00b5L en cada pocillo de la microplaca MANTIS que contiene la muestra. 5. Mezclar. 6. Incubar a temperatura ambiente durante 2 minutos. 7. Colocar la microplaca sobre el MANTIS. 7. Colocar la microplaca sobre el im\u00e1n. 8. Colocar la microplaca sobre el im\u00e1n. 8. Incubar durante 2 minutos. 9. Cerrar la microplaca. 9. Transferir manualmente 12,5 \u00b5l de las microesferas a la nueva microplaca.<\/p><p>D. Volumen de aspiraci\u00f3n = volumen dispensado por muestra x n\u00famero de muestras + factor de seguridad 10%. Para vol\u00famenes de aspiraci\u00f3n superiores a 200 \u00b5l, utilice una punta de pipeta de 1000 \u00b5l.<\/p><h4>Resultados.<\/h4><p>La calidad de la biblioteca Nextera se evalu\u00f3 para cada muestra mediante rastreo con el Agilent BioAnalyzer.<\/p><p><img fetchpriority=\"high\" decoding=\"async\" class=\"alignnone size-large wp-image-25670\" src=\"https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/mantis2-1024x534.png\" alt=\"\" width=\"1024\" height=\"534\" srcset=\"https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/mantis2-1024x534.png 1024w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/mantis2-300x156.png 300w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/mantis2-768x401.png 768w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/mantis2-1536x801.png 1536w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/mantis2-2048x1068.png 2048w\" sizes=\"(max-width: 1024px) 100vw, 1024px\" \/><\/p><p><em>Figura 1. Trazado Bio nalyzer de 8 bibliotecas de secuenciaci\u00f3n de nueva generaci\u00f3n preparadas a partir de 8 muestras \u00fanicas de ADNc de c\u00e9lulas neuronales murinas de 1ng.<\/em><\/p><h4>Resumen.<\/h4><p>Seg\u00fan el trazado de datos del BioAnalyzer, la composici\u00f3n de la biblioteca preparada mediante la miniaturizaci\u00f3n de la reacci\u00f3n en un cuarto de volumen es aceptable y comparable a la preparada seg\u00fan el protocolo experimental de volumen completo.<\/p><p>El procesador de l\u00edquidos MANTIS\u00ae de FORMULATRIX\u00ae facilita la miniaturizaci\u00f3n de las reacciones mediante la dosificaci\u00f3n microflu\u00eddica precisa de reactivos en vol\u00famenes tan bajos como 100 nL, y su uso es sencillo, fiable y sin consumibles.<\/p><p>\u00a0<\/p><p>Fuente: @BostonFummerle<\/p><p>Fecha: 10 de febrero de 2022<\/p>\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t<div class=\"elementor-column elementor-col-50 elementor-inner-column elementor-element elementor-element-2387d1f3\" data-id=\"2387d1f3\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap\">\n\t\t\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t<\/div>","protected":false},"excerpt":{"rendered":"<p>\u4f7f\u7528 MANTIS \u00ae \u6db2\u4f53\u5904\u7406\u5668\u7b80\u5316\u5bf9 [&hellip;]<\/p>","protected":false},"author":1,"featured_media":25681,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"default","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center 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center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-opacity":"","overlay-gradient":""}},"footnotes":""},"categories":[37],"tags":[28,18],"class_list":["post-25667","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-application","tag-formulatrix-mantis","tag-formulatrix"],"yoast_head":"<!-- This site is optimized with the Yoast SEO Premium plugin v21.9 (Yoast SEO v22.7) - https:\/\/yoast.com\/wordpress\/plugins\/seo\/ -->\n<title>\u4f7f\u7528 MANTIS \u00ae \u6db2\u4f53\u5904\u7406\u5668\u7b80\u5316\u5bf9\u5c0f\u9f20\u795e\u7ecf\u5143\u7ec6\u80de CDNA \u6837\u54c1\u8fdb\u884c RNA \u6d4b\u5e8f\u7684 NEXTERA XT \u6587\u5e93\u5236\u5907 -<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" 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