The authors would like to thank members of the Yi laboratory especially Zhaojie Zhang for fruitful discussions regarding linker design and ligation efficiencies, as well as the American Cancer Society for supporting this work through a postdoctoral fellowship (#125209) to J.E.L. Research reported in this publication was also supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01AR059697 (to R.Y.) and a research grant from the Linda Crnic Institute for Down Syndrome. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

NOTE: Il est essentiel de maintenir des conditions RNase-free pendant toute la procédure. 1. adénylation de 3 &#39;Linker <ol><li> Diluer l&#39;ADN oligonucléotide conçu pour les réactions 3 &#39;de ligature à 100 um dans la nucléase-free H 2 O. NOTE: L&#39;oligonucléotide doit avoir un &#39;groupe phosphate, un dinucléotide randomisée en 5&#39; 5 extrémité, et un &#39;3 didésoxycytosine. Le groupe 5 &#39;phosphate est une exigence de l&#39;enzyme efficace pour Mth 5&#39; 10 adénylation. Le phosphate peut être ajouté par un pré-traitement de l&#39;oligonucléotide avec une polynucléotide-kinase, ou commandé par la modification directement. Le dinucléotide randomisée à l&#39;extrémité 5 &#39;est important pour minimiser la préférence de l&#39;ARN ligases qui présentent pour certaines extrémité de jonction bout-réactions sur les autres six séquence. La 3 &#39;didésoxycytosine place un élément de blocage à l&#39;extrémité 3&#39; de l&#39;oligonucléotide to inhiber la formation de concatémères-linker lieur au cours des étapes ultérieures de ligature et sert également à bloquer l&#39;extrémité de la molécule miARN-groupe de liaison formé à l&#39;extrémité de l&#39;extrémité 3 &#39;de la 3 réaction de ligature 11. La séquence oligonucléotidique de liaison choisi pour le procédé de miR-Seq impartiale est la suivante: 4 5&#39;PO NNTGGAATTCTCGGGTGCCAAGG-dideoxyC3. </li><li> Monter la réaction suivante d&#39;adénylation: 200 pmol 3 &#39;de l&#39;oligonucléotide lieur, 4 pl 10x 5 &quot;tampon d&#39;ADN adénylation, 4 pi de 1 mM d&#39;ATP, 4 ul Mth ARN ligase, sans nucléase H 2 O à un volume final de 40 ul. </li><li> Incuber la réaction à 65 ° C pendant 1 heure. Mettre fin à la réaction par chauffage à 85 ° C pendant 5 min. </li><li> Précipiter lieur polyadénylé par l&#39;addition de 2,5 volumes d&#39;éthanol à 100% et 1/3 volume de 10 M d&#39;acétate d&#39;ammonium pour éliminer l&#39;ATP résiduel et pour éviter des ligatures imprévus dans les réactions ultérieures. <ol><li> En bref, mélanger le alalcool, des sels, et une solution d&#39;oligo jusqu&#39;à homogénéité, tournant à pleine vitesse dans une microcentrifugeuse (~ 15 000 x g) à 4 ° C pendant 20 min. Laver le culot dans 70% d&#39;éthanol, l&#39;air sec, et remettre en suspension dans le volume approprié de la nucléase-free H 2 O pour donner 50-100 uM d&#39;oligo adénylé. </li></ol></li><li> Confirmer adénylation en exécutant un gel de polyacrylamide à 18%. <ol><li> Préparer le gel en mélangeant les ingrédients suivants ensemble: 6,3 g d&#39;urée, 6,75 ml de 40% d&#39;acrylamide, et de 1,5 ml de TBE 10x. Ajouter H 2 O à 15 ml. </li><li> Mélanger jusqu&#39;à dissolution complète de l&#39;urée et ajouter 150 ul de 10% (p / v) de persulfate d&#39;ammonium (APS) et 15 ul tetramethlyethylenediamine (TEMED). </li><li> Une fois que le gel a mis, avant-course pendant 30 min à 24 V / cm de largeur de gel avec TBE 1x tampon fonctionnant à température ambiante. Une fois 30 min est au ras des puits avec du tampon en cours d&#39;exécution. </li><li> Mélanger 5 pmoles d&#39;oligonucléotides et polyadénylé avec un volume égal de 2x tampon de dénaturation de l&#39;ARN de chargement (95% (v / v) de formamide,0,02% (p / v) de SDS, 0,02% de bleu de bromophénol (p / v), 0,01% (p / v) Xylène Cyanol, EDTA 1,0 mM), de la chaleur brièvement à 75 ° C et refroidi sur de la glace par encliquetage. Distribuer ensemble de l&#39;échantillon dans le puits du gel. Comprennent également un montant identique de contrôle non-adénylé, et les normes de taille de faible poids moléculaire. </li><li> Exécuter le gel à 24 V / cm jusqu&#39;à ce que le bleu de bromophénol est ¾ du chemin vers le bas du gel. Démonter appareil, gel post-tache avec une solution SYBR-or 1x 1x en TBE et de visualiser sur la boîte transilluminateur UV (Figure 2). REMARQUE: Gel purifier l&#39;oligo adénylé d&#39;une manière similaire à celle décrite ci-dessus, si «aucun ARN de départ&quot; commandes se traduisent par une grande quantité de produit de PCR par comparaison à des échantillons avec de l&#39;ARN d&#39;entrée. </li></ol></li></ol> 2. Linker ligature à l&#39;extrémité 3 &#39;de miARN <ol><li> 3 &#39;Ligation <ol><li> Assembler les éléments suivants sur la glace dans l&#39;ordre indiqué: 1,0 ul de 50% (p / v) de PEG 8000, 0,5 &amp;# 956; l 10x tampon d&#39;ARN ligase (500 mM Tris-HCl pH 7,5, 100 mM de MgCl2, 10 mM de DTT), 1,0 pi polyadénylé en 3 &#39;Lieur (100 pM), un inhibiteur de la RNase 0,5 ul, 0,5 à 8,0 ug d&#39;ARN total, 0,5 ul d&#39;ARN ligase de T4 2, sans nucléase H 2 O à un volume final de 5 ul. </li><li> Mélanger la réaction par pipetage de la solution est trop visqueuse pour vortex dû à la forte concentration de PEG 8000. Une fois intimement mélangé, placer la réaction dans un cycleur thermique à couvercle chauffé. Régler le bloc à 16 ° C, et on incube pendant 4 heures. REMARQUE: La réaction peut être réduite jusqu&#39;à un volume final de 20 ul, sans aucune perte d&#39;efficacité de ligature. Réagir pendant 4 heures à température ambiante ou une nuit à 4 ° C pour obtenir l&#39;efficacité de ligature sensiblement identiques. </li></ol></li><li> Gel Purification <ol><li> Après 3 &#39;ligature, mélanger la réaction avec un volume égal de tampon de charge 2x ARN. Chauffer à 70 ° C pendant 5 min, et enclenchez refroidir sur glace. </li&gt; <li> Exécuter l&#39;échantillon sur un gel de Polyacrylamide à 15% pour PAGE purification. <ol><li> Préparation d&#39;un gel de polyacrylamide à 15% contenant 7 M d&#39;urée, d&#39;une manière similaire à celle décrite précédemment à l&#39;étape 1.5. </li><li> Pré-exécuter le gel à 24 V / cm de largeur de gel pendant au moins 30 min. Coupez l&#39;alimentation, rincer les puits, échantillon de charge et gel de course à même tension que pré-terme. </li><li> Chargez l&#39;échantillon et exécuter la page jusqu&#39;à ce que le bleu de bromophénol vient juste de sortir le gel. Le produit désiré va migrer à proximité de la cyanol de xylène. </li></ol></li></ol></li><li> La coloration, la visualisation et l&#39;excision <ol><li> Démonter appareil de gel et gel de lieu dans le bac propre avec 1x courir tampon (TBE), 1x SYBR-or, et le rock pendant 15 min. </li><li> Retirer le gel de plat coloration et de visualiser sur la boîte de transilluminateur UV recouvert d&#39;une pellicule de plastique propre. Le produit de ligature doit être d&#39;environ 45 nucleotides de longueur. </li><li> Exciser la région contenant le produit de ligature avec un Razo proprelame de r et dans 0,5 ml tube de micro-centrifugeuse. </li></ol></li><li> Isolement du produit et des précipitations <ol><li> Pierce bas de 0,5 ml Tube à centrifuger avec une aiguille 30 G et placer le tube percé dans un second propre, à faible rétention, 1,5 ml microtube. Centrifugeuse pendant 5 min à ~ 15 000 x g. </li><li> Visuellement confirmer que la tranche de gel entière est passée à travers le trou dans le tube intérieur. Si nécessaire, utilisez une pointe de pipette propre à pousser certains fragments de gel plus près du trou. </li><li> Jeter le tube vide de 0,5 ml et ajouter 400 ul de tampon de colonne riche en sel (NaCl 400 mM HSCB, Tris-HCl 25 mM, pH 7,5, 0,1% de SDS) à la suspension de l&#39;acrylamide. tube (s) de Rock à 4 ° C pendant la nuit. </li><li> Transfert suspension à une colonne d&#39;acétate de spin de 0,22 micron de cellulose à l&#39;aide d&#39;une pipette de gros calibre. Centrifuger pendant 3 min à la température ambiante à ~ 15 000 x g pour recueillir tout liquide loin de la matrice de gel d&#39;acrylamide. </li><li> Transférer le liquide à une faible rétention de nettoyage, le tube de 1,5 ml contienttion 1 ul de 20 mg / ml de glycogène. Ajouter un volume égal d&#39;isopropanol et 1/10 volume d&#39;acétate de sodium. Mélanger la solution par inversion de tubes à plusieurs reprises avec le précipité dans le congélateur à -80 ° C pendant 20 min. </li><li> Centrifugeuse à pleine vitesse à 4 ° C pendant 20 min pour sédimenter l&#39;acide nucléique. Retirer le surnageant et laver à l&#39;éthanol à 70%. Air culot sec pendant 10 min à température ambiante et procéder directement à 5 &#39;étape de ligature. </li></ol></li></ol> 3. Linker ligature à extrémité 5 &#39;de miARN-3&#39; Linker hybride <ol><li> Pour un acide nucléique granulés, ajouter les composants suivants: 2 ul de PEG 8000 (50% p / v), 0,5 pi de tampon de ligase ARN 10x, 0,5 pi d&#39;ATP (10 mM), 0,5 pi de N, N-ARN oligo (100 uM), et H 2 O pour un volume final de 4 pl. REMARQUE: La composition de la &#39;NN-ARN oligo&#39; est comme suit 5&#39;-didésoxy-TCUACAGUCCGACGAUCNN3 inversé &quot;et a été purifié par HPLC par le fabricant. </li><li> Mélangercet échantillon par aspiration et à plusieurs reprises vers le bas. Pour ce faire, pendant un certain temps (jusqu&#39;à 5 min) pour s&#39;assurer que la pastille a été complètement re-solubilisé. Si re-solubilisation est problématique, faire chauffer l&#39;échantillon brièvement (3-5 min) à 37 ° C et reprendre pipetage pour mélanger. </li><li> Une fois la pastille est de retour dans une solution, transférer le contenu d&#39;un tube de PCR propre contenant 1 ul d&#39;un mélange 1: 1 d&#39;inhibiteur de RNase et T4 ARN ligase 1 (5 unités). Encore une fois mélanger par aspiration et refoulement. </li><li> Incuber la réaction dans un thermocycleur à 37 ° C pendant 4 heures. Procéder immédiatement à la synthèse de l&#39;ADNc. </li></ol> 4. Transcription inverse / de synthèse d&#39;ADNc <ol><li> Ajouter les éléments suivants à l&#39;extrémité 5 &#39;la réaction de liaison: 2 pl de tampon 5x RT, 0,75 ul de 100 mM de DTT, 1 pi Illumina RT amorce 5&#39;-GCCTTGGCACCCGAGAATTCCA-3&#39; (1 uM), et 0,5 dNTP ul (10 mM). </li><li> Mélanger par pipetage et de la chaleur à 65 ° C pendant 5 min. Puis refroidir lentement sur le banc top. </li><li> Une fois que la réaction est refroidie à la température ambiante, placer le tube sur la glace et ajouter 0,5 pi de chaque enzyme de la transcriptase inverse et un inhibiteur de RNase. Mélanger par pipetage et dans cycleur thermique pour l&#39;étape d&#39;extension est indiqué dans les instructions du fabricant. </li><li> Après ajout total de 5 ul sans nucléase H 2 O pour amener le volume final de la banque d&#39;ADNc à 15 ul. </li></ol> 5. Bibliothèque Préparation <ol><li> Pour l&#39;indexation PCR, assembler la réaction suivante sur la glace: 6 pi de 5x Phusion tampon, une dNTP ul (10 mM), 0,9 pi de DMSO, 0,75 pl amorce 5 &#39;(25 pM), 0,75 pl amorce 3&#39; (25 pM), 3 pl de bibliothèque d&#39;ADNc (concentration variable en fonction du montant de l&#39;entrée), 0,5 pi de polymerase Phusion (1 unité), sans nucléase H 2 O à un volume final de 30 ul. Des séquences d&#39;amorce à assurer la compatibilité avec la matrice et la plate-forme de séquençagechoix. <ol><li> Exécuter la réaction de PCR avec les paramètres suivants: dénaturation initiale à 98 ° C pendant 3 min, pour les 4 premiers cycles, dénaturation à 98 ° C pendant 15 sec, recuit à 50 ° C pendant 15 s, extension à 72 ° C pendant 15 sec . REMARQUE: En fonction de la quantité de la matière d&#39;entrée, varier le nombre total de cycles de PCR. Typiquement, pour une entrée de 1-2 pg d&#39;ARN total, 13 cycles de PCR totaux sont suffisantes pour générer la bibliothèque miR-Seq. </li><li> Effectuer des cycles ultérieurs, par exemple des cycles de 5 à 12, de manière à deux étapes où les cycles de recuit et d&#39;extension sont combinées et réalisées à 70 ° C pendant 30 sec et la dénaturation est de nouveau à 98 ° C pendant 15 s. REMARQUE: Le volume de réaction est de 30 pi pour permettre une pause le programme PCR et supprimer une aliquote de 10 pi au nombre de cycles pré-déterminé à suivre pour l&#39;apparence de produit désiré. En règle générale, les cycles 10, 13, et 16 sont choisis pour l&#39;essai d&#39;un échantillon d&#39;ARN total. </li></ol></li><li> Préparerun gel non dénaturant d&#39;acrylamide à 8% en mélangeant 2 ml de 40% d&#39;acrylamide, 1 ml de TBE 10X, et H 2 O à 10 ml. Puis ajouter 100 ul de 10% d&#39;APS et 10 pi de TEMED. Verser gel et laisser l&#39;ensemble. <ol><li> Pré-exécuter le gel à 12 V / cm de largeur de gel pendant 30 min. Rincer les puits. Ajouter 1 pi de 10 x tampon de charge (15% de Ficoll-400 mM, EDTA 66 20 mM Tris-HCl pH 8,0, 0,1% (p / v) de SDS, 0,01% (p / bleu v) de bromophénol), ajouter 10 ul de échantillon dans chaque puits de dimensions 5 mm x 1 mm x 15 mm. Typiquement, chaque bien peut charger jusqu&#39;à 30 pi d&#39;échantillons. Exécuter le gel de l&#39;échantillon à 10 V / cm de largeur sur gel dans du tampon TBE 1x jusqu&#39;à ce que le colorant bleu de bromophénol passe juste en dehors de la partie inférieure du gel. </li><li> Colorer et visualiser gel comme décrit dans l&#39;étape 2.3. </li><li> L&#39;excision de la bande de 146 paires de base et éluer la nuit dans 0,25 ml de HSCB d&#39;une manière similaire à ce qui est décrit dans la section 2.4. Précipiter l&#39;ADN dans un volume égal d&#39;isopropanol et 1/10 volume d&#39;acétate de sodium. Laver le culot dans 70% eTHANOL et remettre en suspension dans un tampon TE. </li><li> Quantifier la bibliothèque à l&#39;aide PicoGreen selon les recommandations du fabricant. </li></ol></li></ol>

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The authors declare no competing financial interests.

La méthodologie décrite ici fait usage de plusieurs variables clés pour maximiser l&#39;efficacité de la ligature, à savoir de fortes concentrations de PEG, l&#39;utilisation de linkers randomisés, et une forte concentration de linkers 2,6,9. Cette approche permet aux bibliothèques de séquençage fiable quantitatives à partir d&#39;échantillons d&#39;ARN total 2. Nous avons effectué plusieurs titrages d&#39;ARN d&#39;entrée et avons conclu que la méthode précédente est le mieux adapt...

Méthodologies basées séquençage haut-débit ont été largement appliquées à de nombreux échantillons biologiques au cours des dernières années d&#39;expansion grandement notre compréhension de la complexité moléculaire des systèmes biologiques 3,4. Cependant, la préparation des échantillons d&#39;ARN pour le séquençage à haut débit donne souvent des biais spécifiques inhérentes à la méthodologie employée, ce qui limite l&#39;utilité potentielle de ces techniques puissantes. Ces méthodes spécifiques biais ont été bien documentés pour les préparations à base de bibliothèque ligature, petit-ARN 1,2,5,6. Ces préjugés se traduisent par 1000 fois la variation de lit nombre de miARN synthétiques équimolaires, ce qui rend l&#39;inférence de miARN abondance de données de séquençage sauvagement variable et sujette à l&#39;erreur. Les études portant sur ​​les propriétés de la T4 ligase d&#39;ARN de phages dérivés ont démontré que les enzymes présentent préférences fondées nucléotides 7, qui se manifestent bibliothèques comme biaisés dans des expériences de séquençage à haut débit <sjusqu&#39;à&gt; 1,2,8. Afin de minimiser les distorsions conférées par des ligases d&#39;ARN, plusieurs stratégies ont été employées; macromoléculaire encombrement 9, randomiser la séquence nucléotidique de l&#39;adaptateur qui est à proximité du site de ligature 6, et en utilisant des concentrations élevées de ligature adaptateur 2. Grâce à une combinaison de ces trois approches, nous avons développé un flux de travail pour la préparation impartiale de petites bibliothèques d&#39;ARN compatibles pour le séquençage à haut débit (Figure 1). Pour les comparaisons directes entre les protocoles actuels et notre méthode optimisée, se il vous plaît vous référer à notre récent rapport 2. Cette méthode donne des efficacités optimisé de ligature de plus de 95% à la fois en 3 &#39;et 5&#39; et des mesures permettant la ligature sans biais de petites molécules d&#39;ARN à partir d&#39;échantillons biologiques et de synthèse 2.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name of Reagent/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:280px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">3&#39; Linker (5&#39; phosphorylated, 3&#39; blocked)</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">5&#39; Linker</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>5&#39; blocked, HPLC Purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">T4RNL2 (1-249 K227Q)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0351S</td>
			<td>Specialized for ligation of pre-adenylated DNA adapters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">10X Ligation Buffer (without ATP)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0351S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">10X Ligation Buffer (with ATP)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">RNaseOUT&#160;</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">10777-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Polyethylene Glycol (mol. Wt. 8000)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Nuclease-free water</td>
			<td style="width:175px;">Ambion</td>
			<td style="width:157px;">AM9937</td>
			<td>We have found water collected from a distillation apparatus to be of equvalent quality.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">T4RNL1</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Superscript III RT kit</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">18080-051&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Phusion PCR kit</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0530S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina RP1 Primer</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina RT Primer</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina Index Primer(s)</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">40% Acrylamide</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:157px;">BP14081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Urea</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">U6504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Ammonium persulfate</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tetramethyethylenediamine (TEMED)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">2X Denaturing RNA loading buffer</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0351S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Razor blades</td>
			<td style="width:175px;">VWR</td>
			<td style="width:157px;">55411-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">SpinX Centricon Tubes</td>
			<td style="width:175px;">Costar</td>
			<td style="width:157px;">CLS8161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Low Retention Microfuge tubes</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:157px;">02-681-320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sybr Gold</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">S-11494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Adenylation Kit</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">E2610L</td>
			<td></td>
		</tr>
	</tbody>
</table>

highly efficient ligation of small rna molecules for microrna quantitation by high-throughput sequencing

Les miARN clonage et le séquençage à haut débit, appelés miR-Seq, représente à elle seule une approche transcriptome à l&#39;échelle de quantifier miARN avec une résolution d&#39;un seul nucléotide. Cette technique de fixation par capture miARN adaptateurs 3 &#39;et 5&#39; des molécules oligonucléotidiques miARN et permet la découverte de novo miARN. Couplage avec puissantes plates-formes de séquençage de nouvelle génération, miR-Seq a joué un rôle dans l&#39;étude de la biologie des miARN. Cependant, des biais importants introduits par oligonucléotides étapes de ligature ont empêché miR-Seq d&#39;être utilisé comme un outil de quantification précise. Des études antérieures montrent que les biais dans les méthodes miR-Seq actuelles conduisent souvent à inexacts quantification miRNA avec des erreurs jusqu&#39;à 1000 fois pour certains miARN 1,2. Pour résoudre ces biais conférées par la ligature de l&#39;ARN, nous avons mis au point une méthode de ligature petit ARN qui conduit à l&#39;efficacité de ligature de plus de 95% pour les 3 &#39;et 5&#39; des étapes de ligature. Analyse comparative de cette improuvé méthode de construction de bibliothèques utilisant miARN synthétiques équimolaires ou différentielle mixtes, donne constamment lit numéros avec un écart de moins de deux fois de la valeur attendue. En outre, cette haute efficacité méthode miR-Seq permet précise l&#39;échelle du génome miRNA profilage à partir d&#39;échantillons d&#39;ARN totaux in vivo 2.

Les résultats attendus pour la méthode précédente doivent d&#39;abord être l&#39;observation d&#39;un seul changement de nucléotide (augmentation) de la taille de l&#39;oligonucléotide d&#39;ADN qui a été soumis à adénylation par Mth ARN ligase (Figure 2). Suite à 3 &#39;ligation, la visualisation du gel d&#39;acrylamide montre (voir figure 3) des bandes de poids moléculaire élevé tranchants de poids évidents dans la région de 100 à 300 nucléotides du gel. Ce...

Watch this Scientific Journal Video about Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing at JoVE.com

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

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

La ligature très efficace de petites molécules d&#39;ARN pour microARN quantification par séquençage à haut débit

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

highly-efficient-ligation-small-rna-molecules-for-microrna

Research

JoVE Journal

Biology

11.8K vues.  University of Colorado, Boulder. MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs. 

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

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

Gramicidine basée Assay fluorescence; pour déterminer le potentiel de petites molécules pour modifier les propriétés bicouche lipidique

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

Recherche

Biologie

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

Criblage à haut débit pour les petites molécules modulateurs de canaux potassiques entrants Rectifier

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

Génération substrat pour des endonucléases de CRISPR / Cas Systems

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

ARN prédiction de la structure secondaire via SHAPE à haut débit

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

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

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

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

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Génération de haute qualité Immunoprécipitation de la chromatine Patron de l&#39;ADN pour le séquençage à haut débit (ChIP-seq)

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

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Un petit écran Molecule Manuel approchant à haut débit utilisant embryons de poissons zèbres

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Haut-débit criblage de modulateurs chimique des gènes post-transcriptionnelle réglementées

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Automated Modular High Throughput exopolysaccharide plateforme de criblage Couplé avec très sensible Analyse Glucides d&#39;empreintes digitales

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

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

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

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

DamID-seq: L&#39;échelle du génome Cartographie des protéine-ADN Interactions par séquençage à haut débit de fragments d&#39;ADN adénine-méthylé

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

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

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

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

Un protocole pour l&#39;évaluation fonctionnelle de Whole-Protein Saturation mutagenèse Bibliothèques Utilisant à haut débit de séquençage

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