私たちは彼女のサポートと有益なトラブルシューティングの議論に参加するため、特に全体のシグマライフサイエンスミッションのターゲットIDライブラリの開発チームは、技術を発見し、ライセンス条件を交渉するためのケビンGutshallと、ヘザーHolemonに感謝します。我々はまた、自由にそれがクローニングされ得ることに大きなcDNAのバッチとその持続性を準備するために未発表の結果と提案、RxBiosciencesのQaziハミドを共有するためにロンドン大学キングスカレッジの博士ヨープGäkenに感謝します。我々は、cDNA配列とデータ解析を行うためのSAFCからナン·リンスコット·バハルに感謝します。 ミッションは、シグマアルドリッチバイオテクノロジーLPの登録商標です。

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estradiol+downregulates+miR-21+expression+and+increases+miR-21+target+gene+expression+in+MCF-7+breast+cancer+cells.">Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells.</a> Nucleic Acid Res. 37 (8), 2584-2595 .</li><li>Ciaudo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+dynamic+and+sex-specific+expression+of+microRNAs+during+early+ES+cell+differentiation.">Highly dynamic and sex-specific expression of microRNAs during early ES cell differentiation.</a> PLoS Genetics. 5, e1000620 (2009).</li><li>Vallejo, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Notch+signaling+by+the+conserved+miR-8/200+microRNA+family+in+development+and+cancer+cells.">Targeting Notch signaling by the conserved miR-8/200 microRNA family in development and cancer cells.</a> EMBO J. 30 (4), 756-769 (2011).</li><li>Jafarifar, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repression+of+VEGFA+by+CA-rich+element-binding+microRNAs+is+modulated+by+hnRNAP.">Repression of VEGFA by CA-rich element-binding microRNAs is modulated by hnRNAP.</a> L. EMBO J. 30 (7), 1324-1334 (2011).</li><li>Clancy, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+isoform+diversity+can+obscure+detection+of+miRNA-mediated+control+of+translation.">mRNA isoform diversity can obscure detection of miRNA-mediated control of translation.</a> RNA. 17 (6), 1025-1033 (2011).</li></ol>

作者は、提出された作品に関心を持っている営利団体と金銭的関係を宣言します。

マイクロRNAは、mRNAの翻訳を阻害することにより転写後に遺伝子発現を調節すると、頻繁に、標的mRNAを（[6]日）不安定に20から24ヌクレオチドのRNAである。単一のmiRNAは、開発と環境シグナルに対する細胞の応答を制御するために数百のmRNAを調節することができます。標的mRNAを識別し、検証することで、これらの経路におけるmiRNAの役割と機能を決定する上で不可欠である。動物のmiRNAとその標的部位に完全に相補的ではありませんが、しかし、ターゲットの同定は簡単ではありません。 &quot;シード&quot;領域は、miRNAの5 &#39;末端から2〜7を基づいており、通常、そのターゲットに相補的である。しかし、そこに種子のルールには多くの例外があり、塩基対下流には、不完全種子の一致を補正することができます。コンピュータアルゴリズムの数は、シード·マッチングと下流の補償、ターゲット構造に基づいてmiRNAのターゲットを予測するために開発されているDポジション、シーケンスの保全、実験的に検証ターゲット（[7]で検討）のために観察されている他のさまざまなパラメータを設定します。インシリコ予測に便利であると識別多くの有効なmiRNAのターゲットを実行しながら、ほとんどの遺伝子は実験的な検証テストに失敗すると予測し、多くの実際の目標は、予測されていません。コンピュータのアルゴリズムは以前に決定され、ターゲットの機能に基づいているためさらに、彼らは既に知られているものから逸脱してターゲットの検出を許可しません。 実験的なシステムの数が生きている細胞内で機能的なmiRNAのターゲットを識別または発見するために首尾よく使用されています。これらのグローバルなスクリーニング法は、マイクロアレイとRNA配列、RNAの共免疫沈降法（RIP）、および細胞培養におけるアミノ酸と安定同位体標識（SILAC）、プロテオミクスの方法（[7]件）が含まれています。各メソッドには、長所と短所があります。標的miRNAはしばしばmRNAを不安定化し、その劣化、損失oにつながるので、それぞれのmiRNAを模倣または阻害剤を導入した後、mRNAレベルでのRゲインは、miRNAのターゲットを識別することができます。 mRNAのこの損失または利得を容易にマイクロアレイまたはディープシークエンシングにより検出されます。 mRNAの検出方法が簡素化され、タンパク質の検出法よりも高感度であるが、mRNAの検出が劣化しない任意のmiRNAのターゲットを欠場する。 SILACからのもの[8]またはリボソームプロファイリングでのRNA検出からmiRNAのターゲットの結果を比較するバーテルラボからの最近のレポート[9]は、哺乳動物のmiRNAが主にターゲットのmRNAレベルを減少させることによって行動することを示しています。ただし、個々のmiRNAのターゲットでの作業、他の多くのラボでは、蛋白質の変化がmRNAレベルの変化なしを検出した。検出可能なmRNAの損失と翻訳調節と思われるものの報告は、次のとおりです。のmiR-10bがPDCD4でHOXD10 [10]、p27kip1にmiR221/222 [11]、miR-21のp85β[13] [12]は、miR-126を、SIRT1上でのmiR-34 [14] PTEN [15]、のmiR - 中継台Arid4bの[16]は、miR-200C JAG1の[17]、およびmiR-299、297、567、上に、miR-21のVEGFA上のND 609 [18]。また、クランシーら[19]最近のlet - 7の標的部位を含む個々のmRNAアイソフォームが選択的に検出されたときにlet-7による翻訳調節にのみ検出されたことを報告した。すべてのmRNAアイソフォームが1つのmRNAとして検出されたときにそれがある - - として多くのマイクロアレイおよび塩基配列の解析で得られた後者は、少なくともいくつかのケースでは、並進規制が複合プロファイルに見落とされる可能性があることを示唆している。アルゴノート（通常は新生血管傷害）または他の関連タンパク質（RNA免疫沈降、またはRIP）を介してのmiRNA-mRNAの複合体の共免疫沈降に関係なく、調節機構のmiRNAターゲットを分離します。さらに、RIPは、相互作用を内在し、おそらく生物学的に関連が検出されます。しかし、それはすべてのmiRNA-mRNAの相互作用が機能的であり、RIPだけ一時的に関連付けたり、急速に劣化しているすべてのmRNAターゲットを逃すかどうかは知られていない。さらに、特異的なmiRNA-mRNAのパートナーはbioinformatiを推測する必要がありますすべてのmiRNA-mRNAのペアが一緒に共沈自動的ので。最後に、SILACは直接miRNAの調節、タンパク質自体の最終的な製品を識別しますが、小文字は区別されませんので、希少なタンパク質とタンパク質レベルの小さな倍の変化を蹴る。 現在のmiRNAのターゲット同定法の制限の光の中で、我々は別のメカニズムによって機能的なmiRNAのターゲットを識別するための追加のグローバルアッセイが必要になったと感じました。このニーズを満たすために、我々は、ヨープGakenとロンドン大学キングスカレッジのアジムMohamedaliによって発明された技術をライセンスされています。彼らの発明は、二重の選択融合タンパク質であり、具体的には、チミジンキナーゼゼオシン融合、その3&#39;UTRにおける潜在的なmiRNAの標的配列のcDNAライブラリーで規制。 図2に示すように安定TKzeo-cDNA構築物でトランスフェクトした細胞と発現しているが、ゼオシンで選択することができます。興味のあるmiRNAを発現した後、そのmiRNAのの目標は、wiを選択することができます図3に示すように、ガンシクロビル番目。ガンシクロビルは、任意の細胞が関心のmiRNAのターゲットを欠いているTKzeo-cDNAを発現しているチミジンキナーゼを発現する細胞を殺す。標的cDNAは脇腹cDNAおよびPCR産物はターゲットを識別するために配列決定することができますガンシクロビルを使用してプライマーを生き残る細胞からのDNAのPCR増幅により単離することができる。 ミッションのターゲットIDライブラリ - 我々は機能的なヒトmiRNAターゲットのグローバルな識別と発見のための新しいツールにキングス·カレッジ·テクノロジを開発しました。ターゲットIDのライブラリを使用すると、ユーザーは、哺乳動物細胞培養、トランスフェクションと薬剤選択の一連の手順によってmiRNAのターゲットを特定することができます。ライブラリは、包括的であり、ヒト遺伝子の66から79パーセントが含まれています。初期の結果は、両方の前に発見されたほか、新たな目標は、ミッションのターゲットIDライブラリから単離することができることを示しています。 プロトコルは、いくつかの長さですが、哺乳動物細胞培養、トランスフェクション、薬剤選択、PCRおよびシークエンシング - - ターゲットIDのライブラリの使用は標準的な分子生物学研究室の技術を必要とするため、ほとんどの生物学者の手段の範囲内でなければなりません。我々は十分な注意が培養条件を最適化し、適切な実験デザインに支払われ、そして最も重要なのは、ターゲットIDのライブラリと成功を確実にするための選択の手順を実行する（kill-曲線）に最適な薬物濃度を決定するために、各セルの新しいタイプを事前にテストされることを示唆している。 すべてのmiRNAのターゲットの識別方法と同様に、それは非常にターゲットIDのライブラリーをスクリーニングすることによって識別されるターゲットは、マイクロアレイ、定量RT-PCR、レポーターアッセイ（すなわち、ルシフェラーゼ）、またはウェスタン分析などの二番目の方法、使用して検証することをお勧めします。

<table border="1" ><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> JumpStartのREDTaq ReadyMix </td><td> Sigma-Aldrich社</td><td> P0982 </td><td></td></tr><tr><td>ガンシクロビル</td><td> Sigma-Aldrich社</td><td> G2536 </td><td></td></tr><tr><td> G418 </td><td> Sigma-Aldrich社</td><td> G8168 </td><td></td></tr><tr><td>ピューロマイシン</td><td> Sigma-Aldrich社</td><td> P9620 </td><td></td></tr><tr><td> TOPO TA </td><td>インビトロジェン</td><td> KNM4500-01 </td><td></td></tr><tr><td>哺乳類のゲノムDNA MiniprepキットをGenElute </td><td> Sigma-Aldrich社</td><td> G1N10 </td><td></td></tr><tr><td>細胞特異的ヌクレオのkそれ</td><td>ロンザ</td><td></td><td></td></tr><tr><td>のNucleofector </td><td>ロンザ</td><td> AAD-1001 </td><td></td></tr><tr><td>ゼオシン</td><td>インビトロジェン</td><td> <a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=R25001">R250-01</a> </td><td></td></tr><tr><td height="1">ターゲットidライブラリ</td><td> Sigma-Aldrich社</td><td> MREH01 </td><td></td></tr></tbody></table>

genome-wide screen for mirna targets using the mission target id library

ターゲットIDのライブラリは、マイクロRNA（miRNA）のターゲットの検出と識別を支援するために設計されています。ターゲットIDのライブラリは、デュアル選択融合タンパク質を、チミジンキナーゼ-ゼオシン（TKzeo）から3&#39;UTRの下流にクローニングしたプラスミドベースのゲノムワイドなcDNAライブラリーです。選択範囲の最初のラウンドは、安定な形質転換のために興味のあるmiRNAの導入に続いて、最後には、miRNAのターゲットを含むcDNAの選択です。選択されたcDNAは、シークエンシング（ターゲットIDライブラリワークフローと詳細については、図1-3を参照）によって識別されます。 人間のトランスクリプトームの広範なカバレッジを確保するために、ターゲットIDライブラリのcDNAは、複数のヒト組織および細胞株から調製したtotal RNAのプールを使用して、オリゴdTプライミングを経由して生成されました。 1.2キロバイトの平均サイズは、0.5〜4 kbまでのcDNAの範囲を、その結果と（プラスミドマップの図4を参照）p3TKzeoデュアル選択プラスミドにクローニングした。のLiで表される遺伝子標的braryはSigma-AldrichのWebページに記載されています。イルミナシーケンス（ 表3）、の結果から、ライブラリがUCSC RefGeneで21518ユニークな遺伝子の16922（79％）、10または読み出し以上（66％）と14,000の遺伝子が含まれていることを示しています。

Watch this Scientific Journal Video about Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library at JoVE.com

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

ターゲットIDのライブラリは、miRNAのターゲットを識別するために使用されるクローニングされたcDNAのプラスミドベースのゲノムワイドなコレクションです。ここでは、その使用してアプリケーションを示しています。

ミッションターゲットIDのライブラリを使用してmiRNAのターゲットのゲノムワイドスクリーン

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

genome-wide-screen-for-mirna-targets-using-mission-target-id

Research

JoVE Journal

Biology

17.5K ビュー.  Sigma-Aldrich. ターゲットIDのライブラリは、miRNAのターゲットを識別するために使用されるクローニングされたcDNAのプラスミドベースのゲノムワイドなコレクションです。ここでは、その使用してアプリケーションを示しています。

ビデオ: Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

マイクロアレイを用いたtRNAのアミノアシル化（充電）レベルのゲノムワイド解析

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

生物学

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

アイソフォーム特異的遺伝子標的を同定するためのチップを使用してゲノムワイド解析

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

ミッションLentiPlexは、哺乳動物細胞ではshRNAライブラリースクリーニングをプールされた

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

の定量と自動ハイスループットゲノムワイドRNAiスクリーニング C.エレガンス</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of protein-bound DNA and aligning the short reads to a reference genome. Enriched regions are revealed as peaks, which often differ dramatically in shape, depending on the target protein1. For example, transcription factors often bind in a site- and sequence-specific manner and tend to produce punctate peaks, while histone modifications are more pervasive and are characterized by broad, diffuse islands of enrichment2. Reliably identifying these regions was the focus of our work.
Algorithms for analyzing ChIPseq data have employed various methodologies, from heuristics3-5 to more rigorous statistical models, e.g. Hidden Markov Models (HMMs)6-8. We sought a solution that minimized the necessity for difficult-to-define, ad hoc parameters that often compromise resolution and lessen the intuitive usability of the tool. With respect to HMM-based methods, we aimed to curtail parameter estimation procedures and simple, finite state classifications that are often utilized.
Additionally, conventional ChIPseq data analysis involves categorization of the expected read density profiles as either punctate or diffuse followed by subsequent application of the appropriate tool. We further aimed to replace the need for these two distinct models with a single, more versatile model, which can capably address the entire spectrum of data types.
To meet these objectives, we first constructed a statistical framework that naturally modeled ChIPseq data structures using a cutting edge advance in HMMs9, which utilizes only explicit formulas-an innovation crucial to its performance advantages. More sophisticated then heuristic models, our HMM accommodates infinite hidden states through a Bayesian model. We applied it to identifying reasonable change points in read density, which further define segments of enrichment. Our analysis revealed how our Bayesian Change Point (BCP) algorithm had a reduced computational complexity-evidenced by an abridged run time and memory footprint. The BCP algorithm was successfully applied to both punctate peak and diffuse island identification with robust accuracy and limited user-defined parameters. This illustrated both its versatility and ease of use. Consequently, we believe it can be implemented readily across broad ranges of data types and end users in a manner that is easily compared and contrasted, making it a great tool for ChIPseq data analysis that can aid in collaboration and corroboration between research groups. Here, we demonstrate the application of BCP to existing transcription factor10,11 and epigenetic data12 to illustrate its usefulness.

Our Bayesian Change Point (BCP) algorithm builds on state-of-the-art advances in modeling change-points via Hidden Markov Models and applies them to chromatin immunoprecipitation sequencing (ChIPseq) data analysis. BCP performs well in both broad and punctate data types, but excels in accurately identifying robust, reproducible islands of diffuse histone enrichment.

多様ChIPseqデータ型のゲノムワイドな解析のための新規ベイジアン変化点アルゴリズム

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

ゲノムワイドな遺伝子欠失で<em&gt;ストレプトコッカス血統</em&gt;ハイスループットPCRによる

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

3&#39;LIFEアッセイを用いた高スループットにおけるmiRNA標的の検出

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

COSMICと細胞の薬剤-DNA相互作用のゲノムワイドなマッピング（小分子の架橋は、クロマチンを分離するために）

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

真核細胞から体外の環状DNAのゲノムワイドな精製

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

ゲノムワイドな探索とグレープバインで ATL E3 ユビキチンリ ガーゼ遺伝子家族の表現メタ分析のための包括的なワークフロー

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...