我々は化学科学、地球科学とバイオサイエンス部門、基礎エネルギー科学、科学局、米国エネルギー省（受賞番号DE-FG02-91ER20021）と国立科学財団（MCB 0948584）（FB）の事務局のサポートを認める。私たちは、原稿を編集するためのMSカレン鳥に感謝しています。

1。 EMSトリートメント シロイヌナズナの種子は、T /変異が5月7日にC / Gの結果ゲノムのC-to-T変化に誘導される変異剤エチルメタンスルホン酸（EMS）3,4、として使用して変異しています。 <ol><li>細胞小器官蛍光マーカー（具体的には、本研究ではssGFPHDEL（シグナル配列-GFP-HDELテトラペプチド）は、ERのマーカーとして使用されている）を運ぶ0.8グラムシロイヌナズナ種子（〜40,000シーズ）を量る。 </li><li> 50 mlのファルコンチューブに種子を譲渡し、25ミリリットルの蒸留水を追加します。 </li><li> 0.2％（v / v）のエチルメタンスルホン酸塩を追加します。 </li><li>低速でミキサーをnutatingで16時間インキュベートします。 </li><li>液体を吸引除去し、EMSを不活性化するために1.0 M NaOHを含むフラスコにそれを破棄します。 </li><li>近くに、種子を含むファルコンチューブに25mlの水を追加し、5回反転の種子を洗浄するために、すべての種子が定着するまで待機し、その後1.0 Mナトリウムに水を吸引して捨てる<html>フラスコOH。 </li><li>最大10回まで洗浄工程を繰り返します。 </li><li>最後の洗浄後、水の最小量の種子を再懸濁する。 </li><li> 10％の漂白剤の25 mLを加え、シード滅菌に進み、30秒間激しく振る。種子が底に沈殿させ、その後、漂白剤を捨て、滅菌水25 mLで洗浄してください。滅菌水を捨て、25mlの70％エタノールを追加します。 30秒間チューブを振る。種子が底に沈殿しましょう​​。エタノールを捨て、滅菌水25 mLで洗浄してください。滅菌水で2回洗浄を繰り返し、その後3 MMろ紙を含むプラスチック製のペトリ皿に種を注ぎ、ボンネットの下に乾燥させます。 2日間4℃で保管してください。 </li><li> ½MS phytagel（MurashigeおよびSkoog培地の半分の濃度）、150 mmのペトリ皿（プレート〜250から300種）のプレートの種子を。 </li><li>土壌に移植し、2週間プレート上にM1の種子を栽培しています。 </li><li> M2線（1000独立した行）を生成するために、個々のM1植物からM2の種子を収集します。 </li></ol>M2とM3の個体群のove_title &quot;&gt; 2。焦点スクリーニングこのセクションでは、以前の8の説明に従って、共焦点または蛍光顕微鏡で苗の観察を説明します。 <ol><li>各M2ラインから起算して六十種は、プラスチック製のペトリ皿に7〜10日間栽培されている½MS phytagel、同じプレートでも（コントロール）EMS-未処理の5本の苗木を栽培されています。 </li><li> 5〜10子葉は、顕微鏡スライド上にレンズ（40X）に向かって背軸側でマウントし、カバーガラスで囲まれています。 </li><li>それぞれの子葉は、オルガネラのマーカーのいずれかに変更された細胞内分布の蛍光の下で、皮質から内側領域に観察される。 </li><li>陽性植物は、土壌に移植され、その種子はM3世代における変異体の表現型を確認するために再度収集し、スクリーニングされる。 </li><li>野生型ゲノムに少なくとも3回は、おそらく共同戻し交配を通して汚染のバックグラウンドの変異を削除します。希望するオルガネラの蛍光マーカーをntaining。 <ol><li>母植物から、細かいハサミや鉗子を使用すると、成熟したsiliquesを削除して、開いた花。 </li><li>分裂組織から小さすぎるつぼみを削除します。 </li><li> 1花芽を開き、すべての葯を削除するには、花弁とがく片の間に鉗子の一組の先端を挿入します。 </li><li>鉗子のいずれかのペアを使用すると、父親の工場から開いている成熟した花を取ると去勢植物の柱頭に葯をこする。 </li></ol></li></ol> 3。マッピングこのセクションでは、伝統的なマッピング方法10,11と比較する場合に高速ですBorevitz 9時から変更されたプロトコルを使用して、劣性突然変異をマッピングする方法を本質的に説明しています。このアプローチは、単一のアッセイ12に多数の単一の機能多型（SFPを）検出する能力と高密度のオリゴヌクレオチド配列を使用します。アフィメトリクスシロイヌナズナATH1のGeneChipのアレイを使用することも可能です約24,000の遺伝子を分析します。変異を示すF 2個体のプールは、同じ分離集団内で収集された野生型のF 2植物のプールと比較されます。その後、変異は変異体プールが変異遺伝子の対立遺伝子が濃縮されている領域にマッピングされ、その結果、同じ地域に野生型プールは、野生型の親の対立遺伝子13の豊かなことになります。 <ol><li>ゲノムDNA（3μg）をQiagen社DNeasy植物ミニキットを使用して、ホモ接合変異体コロンビア（M3）から抽出され、イルミナGenome AnalyzerのII（GA II）14シークエンシングのために提出されています。 </li><li>ホモ接合変異体コロンビア（M3）は、マッピング集団を生成するためランツベルクエレクタと交配されています。 </li><li>マッピングは、異常な表現型と野生型の表現型とF2植物の同じ数を示す70、最大100 F2個体で実行されます。 </li><li>各工場から穴パンチを使用してリーフディスク（0.60 mm）を収集します。ザリーフディスクは、DNAの類似した量を持っていることを確認するため、同じ年齢の葉から収集する必要があります。サンプルは、個別に、またはグループ内のゲノム抽出のために処理することができます。この場合、それは最終的にはゲノムDNAを抽出する必要があり、そこからより少ないエッペンドルフチュー​​ブを持つようにそれぞれのエッペンドルフチュー​​ブに5〜10サンプルを収集することが可能です。 </li><li> MasterPure植物の葉のDNA精製キット（EPICENTRE）は、ゲノムDNAを抽出するために使用されています。各試料から得られたゲノムDNAは、光度計で定量されています。 </li><li>各サンプルからのゲノムDNAの同量は、植物のDNAを300 ngの（〜30μL）の合計に一緒に入れて、60μL2.5Xランダムプライマー溶液を加え、[125 mMのトリス-HCl（pH6.8）を、12.5mMののMgCl 2、25mMの2 - メルカプトエタノール、750μg/ mlのオリゴデオキシリボヌクレオチドプライマー（ランダム八量体）]（Bioprimeキット）と42μLの水（最終容量132μL）。 </li><li> 5から10分&gt; 95°Cで変性はDNAが。 </li><li>氷の上で涼しい。 </li><li>各denatuへ赤のDNAは、[1 mMのビオチン-14-dCTPを、1mMのdCTPを、2mMのdATPを、2mMのdGTPを、10mMトリス-HCl（pH7.5）で2 mMのdTTPを、1mMのNa2EDTA]ビオチンdCTPで15μL10XのdNTPミックスを追加する（Bioprimeキット）、3μLクレノウポリメラーゼ（Bioprimeキット）で完了します。 </li><li> 25℃で一晩インキュベートする</li><li>ミックス、次の日に、15μlの3 M NaOAc、400μL冷100％エタノールを追加します。 </li><li> 1時間-80℃でインキュベートし、その後15分間20500×gで回転、上清を除去し、500μLの冷75％EtOHで洗浄する。 </li><li> 10分間、20500×gで遠心する。 </li><li> 100μLの水で10分間再懸濁し、37℃で乾燥したDNAペレット。 </li><li>ゲル（ 図2）収量と品質を確認するために5μLを使用しています。 </li><li>のために、野生型と変異型のGeneChip ATH1シロイヌナズナゲノムアレイハイブリダイゼーション反応をラベリングの95μLを送信します。 </li><li> Rソフトウェア（使用してアレイがスキャンされ、得られた。CELファイルが解析され<a href="http://www.r-project.org/">http://www.r-project.oを</a>RG /）。 </li><li> Rのソフトウェアをインストールし、プログラムを開き、次の文字列を貼り付けます。 ソース（ &quot; <a href="http://bioconductor.org/biocLite.R">http://bioconductor.org/biocLite.R</a> &quot;） biocLite（） その後、リターンキーを押して、これは標準BioConductorのパッケージ（インストールされます<a href="http://www.bioconductor.org">http://www.bioconductor.org</a> ）。 </li><li>デスクトップ上の新しいフォルダには、次のWebサイト（からダウンロード<a href="http://www.naturalvariation.org/methods">http://www.naturalvariation.org/methods</a> ）ファイル： readcel.R SFP.R Map.R ath1V5.RData ColLerCEL.zip ファイルColLerCEL.zipが解凍され、結果のファイルは新しいフォルダに貼り付ける必要があります。 </li><li> wildtype.CELとmutant.CELにあなたのGeneChipの実験から得られたデータの名前を変更します。 </li><li>あなたのGeneChipのデータ（wildtype.CELは 、今コピーします。あなたのフォルダにmutant.CEL）。 </li><li>クリックして &quot;その他&quot;とクリックします。オープンR、Mac用） &quot;作業ディレクトリを変更します。&quot;クリックして &quot;ファイル&quot;から &quot;チェンジディレクトリ&quot;：B）PC用</li><li> Macの場合）：ファイルを含むフォルダを選択します。 &quot;ワークスペース&quot;、 &quot;ロード·ワークスペース·ファイルをクリックします。、ファイルを&quot; PC用を選択しath1V5.RData、b）に上記のファイルを含むフォルダを選択し、クリックして&quot;&quot;それから&quot;ロードワーク&quot;とath1V5.RDataを選択します 。 </li><li>メモ帳を使用してreadCEL.Rを開いて、Rにテキスト全体をコピーする</li><li>メモ帳を使用してSFP.Rを開いて、Rにテキスト全体をコピーする</li><li>メモ帳を使用してMap.Rを開いて、Rにテキスト全体をコピーする</li><li>コンソールウィンドウに次のメッセージが表示されます。 X染色体は、MB YZを制限する このリンクを貼り付けTAIRで検索遺伝子 3 <a href="http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y&amp;end=z">http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y</a>＆最後= Z </li><li>インターネットブラウザにリンクをコピーして貼り付けます。ウィンドウが表示され、ファイルを開くか保存するかを尋ねてきます。あなたは、保存を選択する必要があります。ファイル名を選択し、[保存]をクリックし、 &quot;。XLS&quot;拡張子を添付してください。 </li><li>ファイルを開き、あなたの変異体の座標を（結果はグラフィカルに、図3に表される）を見つけることができます。 &quot;XLS&quot;。 </li><li>組み立てイルミナにマップされた領域内の単一塩基多型のうち、特定のEMSの遷移4を識別する変異体ゲノムの読み取り。標準的な手順15を使用して、野生型遺伝子（s）で形質転換して変異体の表現型を補完します。 </li></ol> 4。代表的な結果 図1は、共焦点顕微鏡のスクリーニングを使用して、分泌経路の変異体の同定に使用されるアプローチを示しています。 図2は、アレイハイブリダイゼーションのために標識されたゲノムDNAの典型的な準備を示しています。。 図3に、GeneChipのシロイヌナズナATH1ゲノム配列から得られたデータを分析した後、予想される典型的な結果が提示されます。 <img alt="図1" src="/files/ftp_upload/3809/3809fig1.jpg" /> 図1。ssGFPHDEL（ERマーカー）（1）を発現するシロイヌナズナ形質転換植物は、EMS処理（2）のための十分な種子を生産するために培養した。 EMSで処理した種子は、その後M1植物（3）を生成するために播種した。各M1プラントは別の行を表しており、種子は、それらの各々から別々に収集した。 M2種子は½MS基板（4）上に播種し、共焦点顕微鏡（5）でER形態の欠陥についてスクリーニングした。スクリーニング時に我々は、野生型ERの形態（6）不良ER表現型（7）を示す植物を保存すること植物を発見した。これらの植物は、M3世代を取得し、表現型（8）を確認するために、成長させた。 M3の植物からのゲノムDNAは、Solexaイルミナシーケンス（）を使用した。同じ植物また、F2マッピング集団（b）を得るためにLER-WTとの交雑のために使用されていました。 <img alt="図2" src="/files/ftp_upload/3809/3809fig2.jpg" /> 図2。Bioprimeランダムラベリング反応（100μL、5μL）をアガロースゲル1％ロードされていました。レーンは、野生型F2植物のプールからのものであり、レーンBは変異体F2植物のプールからのものです。 （マーカーはNEBから、1 kbのDNAラダー-N3232）です。 <img alt="図3" src="/files/ftp_upload/3809/3809fig3.jpg" /> 図3図は、GeneChipのシロイヌナズナATH1ゲノムアレイハイブリダイゼーションを使用して、COL-0突然変異のマッピングの例を表しています。この例では変異は染色体1、縦棒で区切られて位置しています。横棒は、検出のしきい値を表しています。

<ol>
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我々は、開示することは何もありません。

ここでは、内膜変異体の同定のための共焦点顕微鏡ベースのスクリーニングを説明しました。このアプローチは、簡単に特定の蛍光タンパク質マーカーが用意されているセルの他の器官に拡張することができます。画面は、ターゲット細胞小器官で、またはマーカーを含むことが想定されていない細胞小器官のいずれかに蛍光マーカーの異常分布を示す変異体の同定に基づいています。それ?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>エチルメタンスルホン酸</td><td>シグマ</td><td> M0880 </td></tr><tr><td> NaOHで</td><td> JTベーカー</td><td> 3722から05 </td></tr><tr><td>ムラシゲスクーグ培地ワットgamborgビタミン</td><td>フィトtechnolog laboratorie </td><td> M404 </td></tr><tr><td> Phytagel </td><td>シグマ</td><td> P8169-1キロ</td></tr><tr><td> RNeasyミニキット</td><td>キアゲン</td><td> 74104 </td></tr><tr><td>マスター純粋な植物の葉のDNA精製キット</td><td> EPICENTRE </td><td> MPP92100 </td></tr><tr><td> Bioprime DNAラベリングシステム</td><td>インビトロジェン</td><td> 18094-011 </td></tr><tr><td>アルコール200プルーフ</td><td>デカンラボラトリーズ。 </td><td> 2716 </td></tr><tr><td> NaOAc </td><td> JTベーカー</td><td></td></tr><tr><td>遺伝子チップシロイヌナズナATH1ゲノム配列</td><td>アフィメトリクス</td><td> 900385 </td></tr><tr><td>ファルコンチューブ50 mLの</td><td>コー​​ニング</td><td> 430290 </td></tr><tr><td>エッペンドルフチュー​​ブを1.5 mL </td><td></td><td></td></tr><tr><td> 90ミリメートルろ紙</td><td>ワットマン</td><td> 1001090 </td></tr><tr><td>化学てんびん</td><td>メトラー·トレドAB54-S </td><td> NA </td></tr><tr><td> Nutating（波）シェーカー</td><td> Heidolph PolyMAXの1040 </td><td> NA </td></tr><tr><td>遠心分離</td><td>エッペンドルフ5417-R </td><td> NA </td></tr></tbody></table>

fluorescence-microscopy screening and next-generation sequencing: useful tools for the identification of genes involved in organelle integrity

このプロトコルは、 シロイヌナズナ実生の蛍光顕微鏡ベースのスクリーニングを説明し、分泌経路内の特定のタグ付き蛍光マーカーの細胞内分布を変化させる劣性変異をマッピングする方法について説明します。 シロイヌナズナたため、そのゲノムサイズの遺伝学的研究のための強力な生物学的なモデルであり、世代時間、王国間の分子機構の保全。分子マーカーに基づいて、従来の方法に代わるもので突然変異をマッピングするためのアプローチとして、ジェノタイピングアレイは、比較的高速であるため有利であると本当に短い時間枠のいくつかの変異体のマッピングを可能にすることができる。この方法は、植物のどのオルガネラの整合性に影響を与えることができるタンパク質の同定を可能にします。ここでは、一例として、我々は小胞体（ER）の整合性のための重要なマップの遺伝子に画面を提案する。我々のアプローチは、しかし、簡単に他の植物の細胞小器官に拡張することができます（例えば、1,2を参照）ため、他の細胞内構造を支配する分子基盤を理解する重要な一歩を表しています。

Watch this Scientific Journal Video about Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity at JoVE.com

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

細胞生物学の基本的なクエストは、真核生物の細胞を作る細胞内小器官のアイデンティティの根底にあるメカニズムを定義することです。ここでは、蛍光顕微鏡と次世代シーケンシングツールを用いた植物オルガネラの形態と機能の整合性の責任遺伝子を同定する手法を提案する。

蛍光顕微鏡スクリーニングと次世代シーケンシング：オルガネラの整合性に関与する遺伝子の同定のための有用なツール

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

fluorescence-microscopy-screening-next-generation-sequencing-useful

Research

JoVE Journal

Biology

12.2K ビュー.  Michigan State University. 細胞生物学の基本的なクエストは、真核生物の細胞を作る細胞内小器官のアイデンティティの根底にあるメカニズムを定義することです。ここでは、蛍光顕微鏡と次世代シーケンシングツールを用いた植物オルガネラの形態と機能の整合性の責任遺伝子を同定する手法を提案する。

ビデオ: Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNAトランスフェクション後の有糸分裂のタイムラプスイメージング

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

生物学

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

線虫における身体サイズ調節に関与する遺伝子をスクリーニングするための戦略を給餌RNA媒介干渉を用いた<em&gt; Cエレガンス</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

16SリボソームRNA遺伝子アンプリコンの次世代シーケンシング

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

次世代シーケンシングによりDNAメチル化解析をターゲット

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

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 ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

レトロウイルス統合サイトの増幅、次世代シーケンシング、およびゲノムDNAのマッピング

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

次世代の 16S rRNA 増幅シーケンスによる目盛りマイクロバイ評価

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

2段階PCRと次世代16S rRNA遺伝子シーケンシングを用いての微生物叢解析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

次世代シーケンシングのための磁気ビーズベースの蚊DNA抽出プロトコル

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.

Methods Article

蛍光顕微鏡スクリーニングと次世代シーケンシング：オルガネラの整合性に関与する遺伝子の同定のための有用なツール