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.

注：それは全体の手順の際にRNaseフリーの条件を維持するために重要である。 3 &#39;リンカーの1アデニル化<ol><li>ヌクレアーゼフリーのH 2 O中に100μMまでの3 &#39;ライゲーション反応のために設計されたDNAオリゴヌクレオチドを希釈 注：オリゴヌクレオチドは、5 &#39;リン酸基、5位の​​無作為化ジヌクレオチド&#39;末端および3 &#39;デオキシシトシンを持つ必要があります。 5アデニル10 &#39;リン酸基は、効率的な5 M番目の酵素の要件である」。リン酸塩は、ポリヌクレオチドキナーゼを有するオリゴヌクレオチドの前処理によって追加された、または直接変更して注文することができる。 5 &#39;末端にランダム化されたヌクレオチドは、特定の最終端が他の6を介して反応を接合するためのRNAリガーゼは、展示順序設定を最小にするために重要である。 3オリゴtの &#39;末端ジデオキシシトシン3にブロッキング要素を配置&#39;Oその後の連結工程の間のリンカー-リンカーコンカテマーの形成を阻害し、ライゲーション反応を11 3 &#39;の端部に形成されたmiRNAのリンカー分子の末端を「ブロックする働きをする。次のように公平なのmiR-配列方法のために選択されたオリゴヌクレオチドリンカー配列は、次のとおりです。5&#39;PO 4 NNTGGAATTCTCGGGTGCCAAGG-dideoxyC3」。 </li><li> 200 pmolの3 &#39;リンカーオリゴヌクレオチド、4μlの10倍の5&#39;のDNAアデニル化バッファー、4μlの1mMのATP、4μlのM番目の RNAリガーゼ、ヌクレアーゼフリーのH 2 O を 40μlの最終体積に次のアデニル化反応を組み立てます。 </li><li> 1時間65℃で反応をインキュベートする。 5分間85℃に加熱して反応を停止。 </li><li>残留ATPを除去し、将来の反応において予期せぬライゲーションを防ぐために、100％エタノールおよび10 M酢酸アンモニウム1/3容積の2.5倍量を添加することによってアデニル化リンカーを沈殿させる。 <ol><li>簡単に言えば、アルを混ぜるcohol、塩、および20分間、4℃で微量フルスピードで回転して均一にオリゴ溶液（〜15000×gで）。アデニル化オリゴの50〜100μMを得るためにヌクレアーゼフリーのH 2 O を適切な容量の70％エタノール、空気乾燥、および再懸濁でペレットを洗浄。 </li></ol></li><li> 18％ポリアクリルアミドゲルを実行することにより、アデニル化を確認します。 <ol><li> 6.3グラムの尿素、40％のアクリルアミドの6.75ミリリットル、および10倍TBEの1.5ミリリットル：一緒に以下を混合することによりゲルを準備します。 H 2 O 15ミリリットルを追加します。 </li><li>尿素が完全に溶解するまで混合し、過硫酸アンモニウム（APS）および15μlのtetramethlyethylenediamine（TEMED）（w / v）の150μlの10％を加える。 </li><li>ゲルを1×TBEを室温でランニング緩衝液でゲル幅の24 V / cm 2で30分間、予備泳動を設定した後。ランニング緩衝液で30分間が起動したらフラッシュ井戸。 </li><li>とアデニル化オリゴヌクレオチドおよびRNAローディングバッファー（95％（v / v）のホルムアミドを、変性2倍の等容積の5ピコモルを混ぜる0.02％SDS、0.02％ブロモフェノールブルー（w / vの）、0.01％（w / vの）（w / v）のキシレンシアノール、1.0mMのEDTA）、熱簡単に75℃、そして氷上で冷却した凍結した。ゲルのウェルにサンプル全体を分注する。また、非アデニル化制御の同一の量、及び低分子量サイズ標準を含む。 </li><li>ブロモフェノールブルーがゲルの底部への道の3/4になるまで、24 V / cmのでゲルを実行します。 1×TBE中の1x SYBR-金溶液と装置、ポストステインゲルを分解し、UV-イルミボックス（ 図2）上で可視化する。 注： &quot;は入力RNA」コントロールは、入力RNAのサンプルと比較して、PCR産物を多量に得られていない場合、ゲルは、上記と同様にアデニル化オリゴを精製する。 </li></ol></li></ol> 2.リンカライゲーションmiRNAの3 &#39;末端に<ol><li> 3 &#39;ライゲーション<ol><li> （w / v）のPEG 8000、0.5＆1.0μlの50％：リストの順に氷上で次のコンポーネントを組み立てる＃956; lの10倍のRNAリガーゼ緩衝液（500mMのトリス-HCl pH7.5で、100mMのMgCl 2を 、10mMのDTT）、1.0μlを、3 &#39;リンカー（100μM）、0.5μlのRNアーゼ阻害剤、0.5〜8.0μgのトータルRNAをアデニル化0.5μlのT4 RNAリガーゼ2、5μlの最終体積にヌクレアーゼフリーのH 2 Oを。 </li><li>解決策が原因で一度完全に混合し、PEG 8000の高濃度の渦に粘性が高すぎるようにピペッティングすることによって反応を混ぜ、加熱蓋付きサーマルサイクラーで反応を置く。 16℃にブロックを設定し、4時間インキュベートする。 注：反応は、ライゲーション効率の損失なしに20μlの最終容量にスケールアップすることができる。本質的に同一のライゲーション効率を得るために、周囲温度でまたは一晩、4℃で4時間反応させる。 </li></ol></li><li>ゲル精製<ol><li> 3 &#39;連結後、2×RNAローディングバッファーを等量の反応混合する。 5分間70℃に加熱し、氷上でクールなスナップ。 </li&gt; <li> PAGE精製のために15％ポリアクリルアミドゲル上でサンプルを実行する。 <ol><li>ステップ1.5で前に説明したのと同様の方法で、7 M尿素を含む15％ポリアクリルアミドゲルを調製。 </li><li>少なくとも30分間ゲル幅の24 V / cmのでゲルを事前に実行します。電源、フラッシュ井戸、負荷サンプルをオフにして、実行前と同じ電圧でゲルを実行します。 </li><li>ブロモフェノールブルーがちょうどゲルを出るまで、サンプルをロードして、ページを実行します。所望の生成物をキシレンシアの近くに移行されます。 </li></ol></li></ol></li><li>染色、可視化、および切除<ol><li> 15分間の1xランニングバッファー（TBE）、1X SYBR-金、岩クリーントレイにゲル装置と場所ゲルを分解します。 </li><li>染色皿からゲルを取り出し、清潔なラップで覆われたUVトランスイルミボックスを視覚化する。ライゲーション産物は、長さが約45ヌクレオチドであるべきである。 </li><li>きれいなrazoでライゲーション産物を含む領域を切り出し0.5ミリリットルのマイクロ遠心管におけるrブレードと場所。 </li></ol></li><li>生成物の単離と降水<ol><li>ピアス30Gの針を0.5 mlマイクロチューブの底と第二クリーンで低リテンションにピアスチューブを配置し、1.5ミリリットルマイクロチューブ。 〜15000×gで5分間遠心する。 </li><li>視覚的に全体のゲル切片は、内管の穴を通って移動したことを確認します。必要であれば、近い穴にいくつかのゲルフラグメントをプッシュするクリーンなピペットチップを使用しています。 </li><li>空の0.5ミリリットルチューブを捨てるとアクリルアミドスラリーに高塩カラムバッファー（HSCB 400のNaCl、25mMのトリス塩酸pH7.5で、0.1％SDS）400μlのを追加します。 4℃で一晩ロック管（群）。 </li><li>ワイドボアピペットチップを用いて0.22ミクロンの酢酸セルローススピンカラムにスラリーを移す。離れアクリルアミドゲルマトリックスからすべての液体を収集する〜15000×gで室温で3分間遠心。 </li><li>クリーンで低保持力に液体を移す、1.5ミリリットルチューブ含める20 mg / mlのグリコーゲンを1μlる。等量のイソプロパノールおよび1/10体積の酢酸ナトリウムを加える。チューブを数回反転させることによって溶液を混合し、20分間℃の冷凍庫-80℃で沈殿する。 </li><li> 20分間、4℃でフルスピードで遠心機は、核酸をペレット化する。上清を除去し、70％エタノールで洗う。室温で10分間空気乾燥したペレットを、5 &#39;ライゲーション工程に直接進む。 </li></ol></li></ol>リンカハイブリッド3.リンカーライゲーション5 &#39;へのmiRNA-3の終わり」 <ol><li>ペレット化核酸に、次のコンポーネントを追加します。2μlのPEG 8000（50％w / v）の、0.5μlの10倍のRNAリガーゼバッファー、0.5μlのATP（10mM）を、0.5μlのNN-RNAオリゴ（100μM）、およびH 4μlの最終体積に2 Oの。 NOTE：5&#39;-反転ジデオキシTCUACAGUCCGACGAUCNN3は以下 &#39;および製造業者によってHPLCで精製したように、「NN-RNAオリゴ&#39;の組成物である。 </li><li>ミックス繰り返し上下にピペッティングすることによって、このサンプル。ペレットが完全に再可溶化されたことを確認するためにいくつかの時間（最大5分）のためにこれを行う。再可溶化は、問題がある場合は、37℃にサンプル短時間（3~5分）を加熱し、混合するためのピペッティングを再開する。 </li><li> RNase阻害剤およびT4 RNAリガーゼ1（5台）の1：1混合ペレットを溶液中に帰ってきたら、1の1μLを含むクリーンなPCRチューブに内容を転送。再び上下にピペッティングして混ぜる。 </li><li> 4時間、37℃でサーマルサイクラーで反応をインキュベートする。 cDNA合成に即座に進みます。 </li></ol> 4.逆転写/ cDNA合成<ol><li> 5に次のコンポーネントを追加 &#39;ライゲーション反応：2μlの5×RT緩衝液、0.75μlの100 mMのDTT、1μlのイルミナのRTプライマー5&#39;-GCCTTGGCACCCGAGAATTCCA-3&#39;（1μM）、および0.5μlのdNTP類（10 mM）を。 </li><li> 5分間65℃にピペッティングし、熱で混ぜる。そして、ゆっくりとベンチtに冷やすオペアンプ。 </li><li>反応物を氷の上で室温、場所管に冷却し、逆転写酵素およびRNase阻害剤のそれぞれ0.5μlを添加したら。メーカーの説明書に示された伸長工程のためのサーマルサイクラー中でピペッティングと場所によって混ぜる。 </li><li>一度15μlの最大のcDNAライブラリの最終容量を持って来るためにヌクレアーゼフリーのH 2 O 5μlを添加完了。 </li></ol> 5.ライブラリの準備<ol><li>インデクシングPCRに、氷上で以下の反応を組み立てる：6μlの5倍のPhusionバッファー、1μlのdNTPを（10 mM）を、0.9μlのDMSO、0.75μlの5 &#39;プライマー（25μM）、0.75μlの3&#39;プライマー（25μM）を、 3μlのcDNAライブラリー（入力の量に応じて可変濃度）、0.5μlののPhusionポリメラーゼ（1単位）、ヌクレアーゼフリーのH 2 O を 30μlの最終体積に。テンプレートとのシーケンシングプラットフォームとの互換性を確保するためのプライマー配列を選択してください選択肢。 <ol><li>最初の4サイクルの間、3分間98℃での初期変性、15秒間98℃の変性、15秒間50℃でのアニーリング、15秒間72℃、延長：以下の設定でPCR反応を実行。 NOTE：入力材料の量に依存して、総PCRサイクル数を変化させる。典型的に、総RNAの1-2μgの入力のために、合計13回のPCRサイクルは、miR-配列のライブラリーを作製するのに十分である。 </li><li>アニーリングおよび伸長のサイクルを合わせて30秒の変性、70℃で実施される二段階の方法で、 例えばその後のサイクル、サイクル5-12を行うには、再び15秒間98℃である。 注：反応容量は、PCRプログラムを一時停止可能にし、所望の生成物の出現をモニターするために予め定められたサイクル数での10μlのアリコートを除去し、30μlのである。典型的には、サイクル10、13、及び16は、全RNA試料からの試験のために選択される。 </li></ol></li><li>準備する40％アクリルアミド2mlの10×TBEを1ml、及びH 2 O 10 mlまで混合することによって、8％非変性アクリルアミドゲル。次いで10％のAPSを100μlとTEMED10μlのを追加します。ゲルを注ぎ、セットしてみましょう。 <ol><li> 30分間のゲル幅の12 V / cmのでゲルを事前に実行します。 ·フラッシュ井戸。 （mMのEDTA、20mMトリス塩酸pH8.0で、0.1％（）​​はw / vの66、15％のフィコール-400、SDS、0.01％（v）のブロモフェノールブルー/ w）の10倍のローディングバッファーの1μlを加え、10μlのを追加寸法×15ミリメートル×1ミリメートル5ミリメートルの各ウェルにサンプル。一般的に、各ウェルは、サンプルの30μlにまで読み込むことができます。ブロモフェノールブルー色素がちょうどゲルの底部がなくなるまで1×TBE緩衝液中のゲル幅の10 V / cmのでサンプルゲルを実行します。 </li><li>ステップ2.3で説明したようにゲルを染色して可視化する。 </li><li> 146塩基対のバンドを切除し、セクション2.4に記載されているものと同様の方法でHSCB 0.25mlの中で一晩溶出する。等量のイソプロパノールおよび1/10体積の酢酸ナトリウムでDNAを沈殿。 70％Eの洗浄ペレットTE緩衝液中thanol再懸濁。 </li><li>製造業者の推奨に従ってピコグリーンを使ってライブラリを定量化する。 </li></ol></li></ol>

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The authors declare no competing financial interests.

本明細書中に記載される方法論は、ライゲーション効率を最大化するためにいくつかの重要な変数の使用、すなわち、高いPEG濃度の、ランダム化されたリンカーの使用、およびリンカー2,6,9-高濃度になる。このアプローチは、総RNAサンプル2から確実に定量的なシーケンシングライブラリーを可能にする。我々は、入力されたRNAの複数の滴定を実施しており、先行する方法は1-...

ハイスループットシークエンシングに基づく方法は、広く大きく、生物系3,4の分子の複雑さの理解を拡大近年、多くの生物学的サンプルに適用されている。しかし、ハイスループット配列決定のためのRNA試料の調製は、多くの場合、これらの強力な技術の潜在的な有用性を制限し、採用手法に固有の固有のバイアスを与える。これらの方法は、特定のバイアスは十分にライゲーションベースの、小RNAライブラリ調製物を1,2,5,6-ために文書化されている。これらのバイアスはがち乱暴変数と誤差シーケンシングデータからのmiRNAの存在量の推定を行う、等モル合成miRNAの番号を読み込み中1000倍のばらつきが生じる。 ファージ由来のT4 RNAリガーゼの特性に焦点を当てた研究では、酵素は、ハイスループットシークエンシング実験においてバイアスされたライブラリとして現れるヌクレオチドベースのプリファレンス7を示すことを報告している<s1,2,8&gt;まで。 RNAリガーゼによって与えられるバイアスを最小にするために、多数の戦略が採用されてきた。高分子、9混雑ライゲーション部位6の近位にあるアダプタのヌクレオチド配列をランダム化し、ライゲーションアダプタ2の高濃度を用いる。これら三つのアプローチの組み合わせを通して、私たちは、ハイスループットシークエンシング（ 図1）のための互換性の低分子RNA図書館に関する調製するためのワークフローを開発しました。現在のプロトコルと私たちの最適化された方法との間の直接の比較のために、私たちの最近の報告書2を参照してください。この最適化された方法は、両方の3 &#39;および5&#39;のステップで95％より高いライゲーション効率が得られ、合成および生物学的試料2からの小RNA分子の公正な連結を可能にする。

<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

のmiRNAのクローニングおよびハイスループットシークエンシング、のmiR-配列と呼ばれるが、一塩基分解能でmiRNAを定量化するためにトランスクリプトーム全体のアプローチとして単独で立っている。この技術は、miRNA分子の3 &#39;および5&#39;オリゴヌクレオチドアダプターを取り付けることによりmiRNAを捕捉し、 デノボ miRNAの検出を可能にする。強力な次世代シーケンシング·プラットフォームとのカップリング、のmiR-配列は、miRNA生物学の研究に尽力してきました。しかし、オリゴヌクレオチドライゲーション手順によって導入の重要なバイアスが正確な定量ツールとして採用されてからは、miR-配列を妨げてきた。これまでの研究では、現在のmiR-のSeq法におけるバイアスはしばしばいくつかのmiRNA 1,2- 1,000倍までのエラーで不正確なmiRNA定量化につながることを実証している。 RNAライゲーションによって与えられるこれらのバイアスを解決するために、我々は両方の3 &#39;および5&#39;ライゲーションステップのため95％以上のライゲーション効率が得られる低分子RNAライゲーション法を開発した。このIMのベンチマーク一貫して期待値から2倍未満偏差の数値を読み取って得られ、等モルまたは差動で混合合成miRNAを使用してライブラリー構築法を証明した。さらに、この高効率のmiR-配列法は、 インビボでの総RNA試料2からの正確なゲノムワイドなmiRNAプロファイリングを可能にする。

先行する方法のため、予想される結果は、最初に第M RNAリガーゼによるアデニル化（ 図2）に供したDNAオリゴヌクレオチドのサイズの単一のヌクレオチドシフト（増加）の観察である必要があります。 3 &#39;ライゲーション後、アクリルアミドゲルの視覚化は、ゲルの100-300ヌクレオチド領域において明らか鋭い高分子量のバンド（ 図3参照）を示している。これ...

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.

ハイスループットシークエンシングによりマイクロRNA定量のための低分子RNA分子の高効率ライゲーション

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 ビュー.  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. 

ビデオ: 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.

グラミシジンベースの蛍光アッセイ、脂質二重層のプロパティの変更の可能性小分子を決定するための

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

生物学

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.

内向き整流カリウムチャネルの小分子モジュレーターのためのハイスループットスクリーニング

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.

CRISPR / CASシステムの酵素のための基質の生成

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.

ハイスループットSHAPEを使用したRNA二次構造予測

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.

ハイスループットシーケンシングのための高品質クロマチン免疫沈降DNAテンプレート（チップ-SEQ）の生成

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.

ゼブラフィッシュ胚を用いた高スループットへのアプローチマニュアル小分子スクリーン

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.

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

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.

自動化されたモジュラーハイスループットエキソポリサッカライドスクリーニングプラットフォームは、高感度炭水化物フィンガープリント分析と相まって

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-配列：アデニンメチル化DNA断片のハイスループットシークエンシングによるタンパク質-DNA相互作用のゲノムワイドなマッピング

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.

ハイスループットシーケンシングを利用し全体プロテイン飽和突然変異誘発ライブラリの機能評価のためのプロトコル

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