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.

Hinweis: Es ist wichtig, RNase-freien Bedingungen während des gesamten Verfahrens aufrechterhalten. 1. Adenylierung 3 &#39;Linker <ol><li> Verdünnte DNA-Oligonukleotid 3 &#39;Ligationsreaktionen bis 100 &amp; mgr; M in Nuklease-freiem H 2 O ausgebildet Hinweis: Das Oligonukleotid sollte eine 5&#39;-Phosphatgruppe, eine randomisierte Dinukleotid am 5&#39;-Ende und ein 3&#39;-Didesoxycytosin haben. Die 5&#39;-Phosphatgruppe ist eine Anforderung des Mth Enzym für effiziente 5&#39;Adenylierungs 10. Das Phosphat kann durch Vorbehandlung des Oligonukleotids mit einer Polynucleotid-Kinase mit der Modifikation direkt zugegeben werden, oder geordnet. Die randomisierte Dinukleotid am 5&#39;-Ende ist wichtig für die Minimierung der Sequenz Bevorzugung, die RNA Ligasen weisen bestimmte End-End-Verbindungsreaktionen über andere 6. Das 3&#39;-Didesoxycytosin stellt ein Sperrelement an dem 3&#39;-Ende des Oligonukleotids to hemmen die Bildung von Linker-Linker Concatemeren während der anschließenden Ligationsschritte und auch dazu dient, das 3&#39;-Ende des miRNA-Linker-Molekül am Ende der 3 &#39;ausgebildet Ligationsreaktion 11 blockieren. Die Oligonukleotid-Linker-Sequenz für die unvoreingenommene miR-Seq Methode gewählt ist wie folgt: 5&#39;PO 4 NNTGGAATTCTCGGGTGCCAAGG-dideoxyC3 &#39;. </li><li> Montieren Sie das folgende Reaktions Adenylierungs: 200 pmol 3&#39;-Linker-Oligonucleotid, 4 ul 10x 5 &#39;DNA Adenylierungs Puffer, 4 &amp; mgr; l 1 mM ATP, 4 &amp; mgr; M-ten RNA-Ligase, Nuklease-freiem H 2 O auf ein Endvolumen von 40 ul. </li><li> Inkubieren Reaktion bei 65 ° C für 1 Stunde. Beenden Reaktion durch Erhitzen auf 85 ° C für 5 min. </li><li> Auszufällen adenylierte Linker durch Zugabe von 2,5 Volumen 100% Ethanol und 1/3 Volumen von 10 M Ammoniumacetat, um Rest ATP zu entfernen und um unvorhergesehene Ligationen in zukünftigen Reaktionen zu vermeiden. <ol><li> Kurz gesagt, mischen die alkohol, Salz und Oligo-Lösung bis zur Homogenität, Spinnen bei voller Geschwindigkeit in einer Mikrozentrifuge (~ 15.000 xg) bei 4 ° C für 20 min. Waschen des Pellets in 70% Ethanol, an der Luft trocknen, und resuspendieren in dem entsprechenden Volumen Nuklease-freies H 2 O auf 50-100 uM adenylierte Oligo ergeben. </li></ol></li><li> Adenylierung bestätigen, indem Sie einen 18% Polyacrylamid-Gel. <ol><li> Vorbereitung des Gels durch Vermischen der folgenden zusammen: 6,3 g Harnstoff, 6,75 ml 40% Acrylamid und 1,5 ml 10x TBE. H In 2 O auf 15 ml. </li><li> Mischen, bis der Harnstoff vollständig gelöst und mit 150 ul 10% (w / v) Ammoniumpersulfat (APS) und 15 ul tetramethlyethylenediamine (TEMED). </li><li> Sobald Gel, Vorlauf für 30 min mit 1x TBE-Laufpuffer bei Raumtemperatur eingestellt, bei 24 V / cm Gel Breite. Nachdem 30 min ist bündig Wells mit Laufpuffer. </li><li> Mischungs 5 ​​pmol der Oligonukleotide mit adenylierte und gleichen Volumen 2x denaturierenden RNA-Ladepuffer (95% (v / v) Formamid,0,02% (w / v) SDS, 0,02% Bromphenolblau (w / v), 0,01% (w / v) Xylencyanol, 1,0 mM EDTA), kurz erhitzen auf 75 ° C und rasten auf Eis gekühlt. Dispense gesamte Probe in die Vertiefung des Gels. Auch eine identische Menge an nicht-adenylierte Kontrolle und niedrige Standards Molekulargewicht Größe. </li><li> Die Elektrophorese bei 24 V / cm, bis das Bromphenolblau auf ¾ der bis zum Boden des Gels. Zerlegen Gerät, post-Fleck-Gel mit 1x SYBR-Gold-Lösung in 1x TBE und visualisieren auf UV-Transilluminator Feld (Abbildung 2). HINWEIS: Gel zur Reinigung des adenylierte oligo in einer ähnlichen Weise wie oben beschrieben, wenn &quot;kein Eingangs RNA&quot; Kontrollen, was zu einer hohen Menge an PCR-Produkt im Vergleich zu Proben, die mit Eingangs RNA. </li></ol></li></ol> 2. Linker-Ligation, um 3&#39;-Ende des miRNA <ol><li> 3 &#39;Die Ligation <ol><li> Montieren Sie folgende Komponenten auf Eis in der aufgeführten Reihenfolge: 1,0 ul 50% (w / v) PEG 8000, 0,5 &amp;# 956; l 10x RNA-Ligase-Puffer (500 mM Tris-HCl pH 7,5, 100 mM MgCl 2, 10 mM DTT), 1,0 ul adenylierte 3&#39;-Linker (100 &amp; mgr; M), 0,5 ul RNase-Inhibitor, 0,5-8,0 &amp; mgr; g Gesamt-RNA, 0,5 &amp; mgr; l T4-RNA-Ligase 2, Nuklease-freiem H 2 O auf ein Endvolumen von 5 &amp; mgr; l. </li><li> Mischen Sie die Reaktion durch Pipettieren, wenn die Lösung zu viskos, um Wirbel aufgrund der hohen Konzentration von PEG 8000. Nachdem gründlich gemischt, legen die Reaktion in einem Thermocycler mit einem beheizbaren Deckel. Stellen Sie den Block auf 16 ° C, und Inkubation für 4 h. HINWEIS: Die Reaktion kann bis zu einem Endvolumen von 20 &amp; mgr; l ohne Verlust der Ligationseffizienz skaliert werden. Reaktion 4 h bei Raumtemperatur oder über Nacht bei 4 ° C, um im wesentlichen identische Ligationseffizienz zu erhalten. </li></ol></li><li> Gel Purification <ol><li> Folgende 3 &#39;Ligation, mischen die Reaktion mit einem gleichen Volumen 2x RNA-Ladepuffer. Hitze bis 70 ° C für 5 min, und Schnapp kühl auf Eis. </li&gt; <li> Führen Sie das Beispiel auf einem 15% Polyacrylamid-Gel für PAGE Reinigung. <ol><li> Vorbereiten einer 15% igen Polyacrylamidgel, enthaltend 7 M Harnstoff, in einer Weise ähnlich zu der zuvor in Schritt 1.5 beschrieben. </li><li> Vorlauf das Gel bei 24 V / cm Gel Breite für mindestens 30 min. Schalten Sie Stromversorgung, bündig Brunnen, Lastprobe und Lauf Gel bei gleichen Spannung wie Vorlauf. </li><li> Laden Sie die Probe und führen Sie den PAGE, bis die Bromphenolblau hat gerade das Gel verlassen. Das gewünschte Produkt migrieren wird in der Nähe des Xylencyanol. </li></ol></li></ol></li><li> Färbung, Visualisierung und Excision <ol><li> Zerlegen Sie Gel-Apparatur und Ort Gel in sauberen Tablett mit 1x Laufpuffer (TBE), 1x SYBR-Gold, und Rock für 15 min. </li><li> Entfernen Gel aus Färbeschale und visualisieren auf UV-Transilluminator Box mit sauberen Plastikfolie abgedeckt. Das Ligationsprodukt sollte etwa 45 Nukleotide lang sein. </li><li> Auszuschneiden die Region mit dem Ligationsprodukt mit einem sauberen razor Klinge und in 0,5 ml Mikrozentrifugenröhrchen. </li></ol></li><li> Produkt Isolierung und Niederschlag <ol><li> Pierce unten von 0,5 ml Mikrozentrifugenröhrchen mit einer 30 G-Nadel und legen Sie die durchbohrten Rohr in einem zweiten sauberen, Low-Retention, 1,5 ml Mikrozentrifugenröhrchen. Zentrifuge 5 min bei ~ 15.000 x g. </li><li> Visuell zu bestätigen, dass die gesamte Gelscheibe hat durch das Loch in der inneren Röhre bewegt. Falls erforderlich, verwenden Sie ein sauberes Pipettenspitze, einige Gelfragmente näher an das Loch zu schieben. </li><li> Entsorgen Sie die leere 0,5-ml-Röhrchen und fügen 400 ul Hochsalzsäulenpuffer (HSCB 400 mM NaCl, 25 mM Tris-HCl pH 7,5, 0,1% SDS) an die Acrylamid Gülle. Rockröhre (n) bei 4 ° C über Nacht. </li><li> Übertragen Aufschlämmung auf einen 0,22 Mikron Zelluloseacetat-Spin-Säule unter Verwendung einer großen Bohrung Pipettenspitze. Zentrifuge für 3 min bei Raumtemperatur bei ~ 15.000 xg, um weg von Acrylamid-Gel-Matrix sammeln alle Flüssigkeit. </li><li> Übertragen Sie die Flüssigkeit auf eine saubere, Low-Retention, 1,5-ml-Röhrchen enthaltening 1 ul 20 mg / ml Glykogen. Hinzufügen eines gleichen Volumens an Isopropanol und 1/10 Volumen Natriumacetat. Mix-Lösung durch Umdrehen des Röhrchens mehrmals dem Niederschlag in -80 ° C Gefrierschrank für 20 min. </li><li> Zentrifuge bei voller Geschwindigkeit bei 4 ° C für 20 min, um die Nukleinsäure zu pelletieren. Überstand entfernen und waschen in 70% Ethanol. Luft trocknen Pellet für 10 min bei Raumtemperatur und direkt zum 5 &#39;Ligationsschritt. </li></ol></li></ol> 3. Linker-Ligation zu 5&#39;-Ende der miRNA-3 &#39;Linker Hybrid <ol><li> Zu pelle Nukleinsäure, fügen die folgenden Komponenten: 2 &amp; mgr; l PEG 8000 (50% w / v), 0,5 ul 10x-RNA-Ligase-Puffer, 0,5 ul ATP (10 mM), 0,5 &amp; mgr; l N, N-RNA-Oligo (100 &amp; mgr; M) und H 2 O auf ein Endvolumen von 4 &amp; mgr; l. HINWEIS: Die Zusammensetzung der &quot;NN-RNA-Oligo&quot; ist wie folgt: 5&#39;-invertierte Didesoxy-TCUACAGUCCGACGAUCNN3 &#39;und wurde von dem Hersteller über HPLC gereinigt. </li><li> Mischendiese Probe durch Auf- und Abpipettieren wiederholt. Dies für einige Zeit (bis zu 5 min), um zu gewährleisten, dass das Pellet wurde vollständig neu solubilisiert worden. Wenn erneute Solubilisierung ist problematisch, erwärmen die Probe kurz (3-5 min) bis 37 ° C und wieder Pipettieren gemischt. </li><li> Sobald das Pellet wieder in Lösung gebracht, wird der Inhalt in einen sauberen PCR-Röhrchen, enthaltend 1 &amp; mgr; l einer 1: 1 Mischung von RNase-Inhibitor und T4 RNA Ligase 1 (5 Einheiten). Wieder mischen und Abpipettieren. </li><li> Inkubieren der Reaktion in einem Thermocycler bei 37 ° C für 4 Std. Gehen Sie sofort zum cDNA-Synthese. </li></ol> 4. Reverse Transkription / cDNA Synthesis <ol><li> Fügen Sie die folgenden Komponenten in den 5 &#39;Ligation-Reaktion: 2 ul 5x RT-Puffer, 0,75 &amp; mgr; l 100 mM DTT, 1 ul Illumina RT Primer 5&#39;-GCCTTGGCACCCGAGAATTCCA-3&#39; (1 &amp; mgr; M) und 0,5 ul dNTPs (10 mM). </li><li> Mischen durch Pipettieren und Wärme auf 65 ° C für 5 min. Dann langsam auf der Bank t abkühlenop. </li><li> Sobald Reaktion auf Raumtemperatur, das Röhrchen auf Eis gekühlt und mit 0,5 &amp; mgr; l jeweils von Reverse Transkriptase und RNase-Inhibitor. Mischen durch Pipettieren und in Thermocycler für die Erweiterung Schritt in den Anweisungen des Herstellers angegeben. </li><li> Sobald vollständige Add 5 ul Nuklease-freies H 2 O, um das Endvolumen der cDNA-Bibliothek bis zu 15 &amp; mgr; l zu bringen. </li></ol> 5. Bibliothek Vorbereitung <ol><li> Zur Indexierung PCR, montieren die folgende Reaktion auf Eis: 6 ul 5x Phusion Puffer, 1 ul dNTPs (10 mM), 0,9 &amp; mgr; l DMSO, 0,75 &amp; mgr; l 5&#39;-Primer (25 uM), 0,75 &amp; mgr; 3 &#39;Primer (25 uM), 3 &amp; mgr; l cDNA-Bibliothek (variabler Konzentration in Abhängigkeit von der Menge der Eingabe), 0,5 ul Phusion Polymerase (1 Einheit), Nuklease-freiem H 2 O auf ein Endvolumen von 30 ul. Wähle die Primer-Sequenzen, um die Kompatibilität mit der Schablone und der Sequenzierungsplattform gewährleistenWahl. <ol><li> Führen Sie die PCR-Reaktion mit den folgenden Einstellungen: anfängliche Denaturierung bei 98 ° C für 3 min, für die ersten 4 Zyklen Denaturierung 98 ° C für 15 sec, Temperung bei 50 ° C für 15 sec, erstrecken sich bei 72 ° C für 15 sec . HINWEIS: Je nach der Menge des Ausgangsmaterials, variieren die Gesamt PCR-Zyklus-Nummern. Typischerweise ist für einen Eingang 1-2 ug Gesamt-RNA, sind insgesamt 13 PCR-Zyklen ausreicht, um die miR-Seq-Bibliothek zu erzeugen. </li><li> Durchführung nachfolgenden Zyklen, beispielsweise Zyklen 5-12, in einem Zwei-Schritt-Weise, wo die Hybridisierung und Verlängerung Zyklen werden vereinigt und bei 70 ° C für 30 sec und der Denaturierung wieder 98 ° C für 15 sec. HINWEIS: Das Reaktionsvolumen beträgt 30 ul Pausieren der PCR-Programm zu ermöglichen, und nehmen Sie die 10 ul Aliquot in vorbestimmten Zykluszahlen für Aussehen des gewünschten Produkts zu überwachen. Typischerweise Zyklen 10, 13 und 16 sind für die Prüfung von einer RNA-Probe ausgewählt. </li></ol></li><li> Vorbereiteneine 8% nicht-denaturierenden Acrylamid-Gel durch Mischen von 2 ml 40% Acrylamid, 1 ml 10x TBE, und H 2 O auf 10 ml. Dann fügen Sie 100 ul 10% APS und 10 ul TEMED. Gießen Gel und lassen Set. <ol><li> Vorlauf das Gel bei 12 V / cm Gel Breite für 30 min. Flush Brunnen. 1 ul 10x-Ladepuffer (15% Ficoll-400, 66 mM EDTA, 20 mM Tris-HCl pH 8,0, 0,1% (w / v) SDS, 0,01% (w / v) Bromphenolblau), mit 10 ul Probe in jede Vertiefung der Abmessungen 5 mm x 1 mm x 15 mm. Typischerweise kann jeder Vertiefung bis zu 30 &amp; mgr; l-Proben zu laden. Ausführen des Muster Gel bei 10 V / cm Gel Breite in 1x TBE-Puffer, bis der Bromphenolblau-Farbstoff läuft gerade aus dem Boden des Gels. </li><li> Färben und Visualisieren Gel wie in Schritt 2.3 beschrieben. </li><li> Verbrauchsteuern Das 146 Basenpaar-Band und eluieren über Nacht in 0,25 ml HSCB in einer Weise ähnlich dem, was in Abschnitt 2.4 beschrieben. Auszufällen DNA in einem gleichen Volumen an Isopropanol und 1/10 Volumen Natriumacetat. Wash Pellet in 70% Ethanol und Resuspension in TE-Puffer. </li><li> Quantifizieren Sie die Bibliothek mit PicoGreen nach den Empfehlungen des Herstellers. </li></ol></li></ol>

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The authors declare no competing financial interests.

Die hierin beschriebene Methode verwendet mehrere Schlüsselvariablen Ligationseffizienzen, nämlich hohe Konzentrationen an PEG, die Verwendung von randomisierten Linker und hohe Konzentration von Linkern 2,6,9 maximieren. Dieser Ansatz ermöglicht zuverlässig quantitative Sequenzierung Bibliotheken aus Gesamt-RNA-Proben 2. Wir haben mehrere Titrationen von Eingangs RNA durchgeführt und dabei festgestellt, dass die vorangehende Methodik eignet sich am besten für die Gesamt-RNA Mengen in der 1-8...

Hochdurchsatz-Sequenzierung basierte Methoden sind weit verbreitet, viele biologische Proben in den letzten Jahren erheblich erweitern unser Verständnis der molekularen Komplexität biologischer Systeme 3,4 angewendet. Allerdings Vorbereitung RNA-Proben für Hochdurchsatz-Sequenzierung oft vermittelt spezifische Vorurteile inhärent angewandten Methode, die Begrenzung der potenziellen Nutzen dieser mächtigen Techniken. Diese Methode bestimmte Vorurteile sind gut für die Ligation-basierten, kleine RNA-Bibliothek Vorbereitungen 1,2,5,6 dokumentiert. Diese Vorspannungen ergeben 1.000-fache Variation liest Nummern äquimolaren synthetische miRNAs, wodurch Inferenz miRNA Fülle von Sequenzdaten wild variable und fehleranfällig. Studien, die sich auf die Eigenschaften des Phagen T4 abgeleitetes RNA Ligasen haben dokumentiert, dass die Enzyme weisen Nucleotid-basierte Präferenzen 7, die als vorgespannte Bibliotheken in Hochdurchsatz-Sequenzierung Experimenten manifestieren <sup&gt; 1,2,8. Um die Vorspannungen von RNA Ligasen verliehen minimieren, verschiedene Strategien eingesetzt wurden; makromolekulare Verdrängung 9, Randomisierung der Nukleotidsequenz des Adapters, die proximal zu der Ligationsstelle 6 ist, und unter Verwendung hoher Konzentrationen von Ligations Adapter 2. Durch eine Kombination dieser drei Ansätze haben wir einen Arbeitsablauf für unvoreingenommene Herstellung von kleinen RNA-Bibliotheken für Hochdurchsatz-Sequenzierung (Abbildung 1) kompatibel entwickelt. Für direkte Vergleiche zwischen aktuellen Protokolle und unser optimiertes Verfahren entnehmen Sie bitte unserem aktuellen Bericht 2 beziehen. Diese optimierte Verfahren ergibt Ligationsgrade von mehr als 95% an beiden 3 &#39;und 5&#39; Schritte und ermöglicht die unvoreingenommene Ligation von kleinen RNA-Molekülen aus synthetischen und biologischen Proben 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

Mirna Klonierung und Hochdurchsatz-Sequenzierung, genannt miR-Seq, steht allein als Transkriptom-weiten Ansatz für miRNAs mit Einzel-Nukleotid-Auflösung zu quantifizieren. Diese Technik nimmt miRNAs durch Anbringen 3 &#39;und 5&#39;-Oligonukleotid-Adapter zu miRNA-Moleküle und können de novo miRNA Entdeckung. Die Kopplung mit leistungsstarken nächsten Generation Sequenzierung Plattformen hat miR-Seq war maßgeblich an der Studie der miRNA Biologie. Allerdings haben erhebliche Verzerrungen durch Oligonukleotid-Ligation Schritte eingeleitet miR-Seq davor als eine genaue Quantifizierung Werkzeug eingesetzt verhindert. Frühere Studien zeigen, dass Vorurteile in aktuellen miR-Seq Methoden oft zu ungenauen miRNA Quantifizierung führen mit Fehlern bis zu 1.000-fach für einige miRNAs 1,2. Um diese Verzerrungen durch RNA-Ligation vermittelt zu lösen, haben wir ein kleines RNA Ligationsverfahren, die in Ligationsgrade von über 95% sowohl in 3 &#39;und 5&#39; Ligationsschritte ergibt entwickelt. Benchmarking diese imbewiesen Bibliothek Bauweise mit äquimolaren oder differentiell gemischten synthetischen miRNAs, konsequent ergibt liest Nummern mit weniger als zweifache Abweichung vom Erwartungswert. Darüber hinaus ermöglicht diese Hocheffizienz miR-Seq Methode genaue genomweiten miRNA Profiling von in vivo Gesamt-RNA-Proben 2.

Die erwarteten Ergebnisse für die vorstehenden Verfahren sollte zunächst Beobachtung einer einzelnen Nukleotid-Verschiebung (Anstieg) in der Größe der DNA-Oligonukleotid, das unter Adenylierung von Mth RNA-Ligase (Abbildung 2). Nach 3 &#39;Ligation, Visualisierung des Acrylamid-Gel zeigt (siehe Abbildung 3) scharfe Gewichtsbändern mit hohem Molekular deutlich in der 100-300 Nukleotidbereich des Gels. Dies zeigt, dass die RNA-Probe verwendet wird ist von hoher Qualität (ni...

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.

Hoch effiziente Ligation kleine RNA-Moleküle für MicroRNA Quantifizierung von Hochdurchsatz-Sequenzierung

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 Aufrufe.  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. 

Serie: 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.

Gramicidin-basierte Fluoreszenz-Assay; zur Bestimmung Small Molecules Potential für das Ändern von Lipid-Doppelschicht-Eigenschaften

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

Forschung

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.

High-throughput Screening niedermolekularer Modulatoren von Inward Gleichrichter-Kaliumkanäle

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.

Substrat Generation für Endonukleasen der CRISPR / Cas-Systeme

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.

RNA Secondary Structure Prediction Mit 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 ...

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.

Generierung von High Quality Chromatin Immunpräzipitation DNA Vorlage für Hochdurchsatz-Sequenzierung (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.

Ein Handbuch Kleine Molecule Bildschirm Nähern Hochdurch Mit Zebrafischembryonen

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.

Hochdurchsatz-Screening für Chemische Modulatoren der Post-transkriptionell regulierten Genen

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.

Automatisierte Modular High Throughput Exopolysaccharid Screening-Plattform mit hochsensiblen Carbohydrate Fingerabdruck-Analyse Gekoppelt

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: Genomweite Mapping von Protein-DNA-Wechselwirkungen von Hochdurchsatz-Sequenzierung von Adenin-methylierten DNA-Fragmenten

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.

Ein Protokoll für Functional Assessment of Whole-Protein Sättigungsmutagenese-Bibliotheken mit Hilfe von 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 ...