著者は、シンガポールのA * STARのサポートされています。さらに、MJFは、科学国立フェローシップとリー·クアンユーポスドクフェローシップの女性のためのA * STAR、国立科学奨学金、ロレアルでサポートされています。 YRはNIH ENCODEの助成金（R01 HG004456-01、R01 HG003521-01）でサポートされています。著者はまた、ビデオ編集およびさんミシェル·テオのために氏ギャラリー一覧ラヒム、シーンを撮影するため、特定の氏ケルビンイッセイ氏、チャン·カイ翔氏とシャーウィンガンの8ピクセルプロダクション、シンガポール、ビデオ撮影チームを認めるボイスオーバー。

<ol>
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利害の衝突が宣言されません。

CHIA-PETは、転写調節における長距離相互作用を識別するために開発した方法です。 CHIA-PETライブラリの品質を決定する重要な要因の一つはChIP材料の品質です。 ビデオに示すように、プロトコルは、セルをクロスリンクするEGSとホルムアルデヒドの使用が組み込まれています。より長いスペーサーアームを有する第二架橋試薬と組み合わせて、ホルムアルデヒドの使用は、単独で3,11,15ホルムアルデヒドによってバインドできませんでしたタンパク質の結合に役立つ可能性があります。我々は、堅牢な結合部位との長距離相互作用9を示しているこの方法でライブラリを構築した。しかし、架橋やチップの条件は、関心のある各要因のために最適化し、あまりにも多くの架橋は、超音波処理により断片化が困難になり、おそらく偽のクロマチンの相互作用が生じる可能性があるので、それは過剰架橋に重要ではありませんする必要があります。クロマチンの相互作用CHIA-PETによって識別されるこのような蛍光in situハイブリダイゼーション4などの別の方法によって検証する必要があります。 我々はクロマチン材料の100 ngの最小をお勧めします。我々はクロマチン材料の50 ngから質の良いライブラリを構築しているが、我々は、出発物質の大量の、それにより、各ライブラリのアンプリコンと冗長性を最小限に抑え、16未満のPCRサイクルでCHIA-PETライブラリーの構築を許可することを観察した。この下の冗長性は、それによってシーケンスの少ない車線の、より包括的なクロマチンの相互作用マップを有効にすると、より高いユニークなマップされたタグも使用可能なデータの割合が高いと相関していた。各チューブのビーズの最終的な詰め量はそれぞれ磁気およびセファロースビーズに50μlと100μlのでなければなりません。パックされたビーズのボリュームが述べたよりも小さい場合、DNA有利子ビーズの損失を最小化するために同様に事前にクリアブランク磁気またはセファロースビーズを用いた最小のパックボリュームにもたらす以降のステップでの。ちびのヒントや大規模なコアチップは、セファロースビーズをピペッティングするために使用されるべきです。 次の変更は、以前公開されCHIA-PETプロトコル5次の組み込まれた。まず、磁気Gビーズを洗浄中に、サンプルの損失を最小限に抑えるために使用された。また、セルフライゲーションハーフリンカーまたは/およびアダプタのアンプリコンであることが100 bpおよび138 bpのおおよそのサイズの非特異的なバンドを同定した。したがって、我々は、PCR増幅の間に非特異的バンドを最小限に抑えるために、ビオチン化半リンカーと454 GS20アダプタの濃度を減少させた。近接ライゲーションボリュームは、その後の精製工程中のサンプルの損失を最小限に抑え、また、試薬コストを節約するために50ミリリットルから10ミリリットルに減少した。また、ストレプトアビジンビーズにCHIA-PET DNAの最大のキャプチャを確保するためにビーズにCHIA-PETのDNAを固定するためにインキュベーション時間の増加となりました。 近接ライゲーションステップの間、キメラライゲーションTHAtは、必然的に非特異的とランダムに生成されたin vivoでクロマチンの相互作用で真を表すものではありません。したがって、任意のCHIA-PET実験からのデータの品質を評価するために、キメラ率が特定の塩基バーコードTAAGとATGT 5を持つ2つの異なる半リンカーの使用から推定される。ハイスループットシーケンシングした後、CHIA-PETシーケンスは、最初の8を区別することができる特定のライゲーション産物と非特異的ライゲーション産物から派生したリンカバーの組成と配列について分析されます。知られているキメラの割合（すなわち、ヘテロABリンカー）は、社内でMCF-7 RNAポリメラーゼII CHIA-PETのライブラリが15％未満である本。 CHIA-PETのシーケンスは、その後、すなわち2つのカテゴリ、セルフライゲーションペットと間ライゲーションのペットに分類されます。間ライゲーションのペットがiから派生している間にセルフライゲーションのペットは、クロマチンの断片の自己環状化ライゲーションから得られる2つの異なるDNA断片の間NTER-ライゲーション。後者は、同じ染色体（染色体内間ライゲーションペット）、あるいは両方のタグが2つの異なる染色体（染色体間の相互連結PETS）にマッピングされ、各タグのゲノム距離に基づいて3つのカテゴリに分割さです。我々は、異なるカテゴリ8を整理するCHIA-PETツールのソフトウェアパッケージを開発しました。これは、ライブラリ内にあるDNA断片に基づいて行われます。一般的には、小さなチップの断片は、より高い解像度を提供し、4キロバイト程度であるこれらのRNAポリメラーゼII CHIA-PETライブラリをカットオフします。 さらに、実際のクロマチンの相互作用は相互作用クラスタ内の間ライゲーションのペットの数をカウントすることによって、ランダムノイズと区別することができます。言い換えれば、高いPETカウントのクラスタは、実際のクロマチンの相互作用8であることの確率が高いと言われています。 私たちの偽陽性をフ​​ィルタリングするには偶然によって間ライゲーションのペットを形成することができる高濃縮アンカーからの上昇は、統計分析のフレームワークは、2つのアンカー8の任意の間ライゲーションのペットのランダムな形成を考慮して策定されています。 結論として、CHIA-PET技術は、地球規模でのクロマチンの相互作用ネットワークをマッピングすることができます。 CHIA-PETでのチップの実装では、ライブラリの複雑さとバックグラウンドノイズの低減を可能にします。さらに、チップは、特定の転写因子5に関連付けられた特定のクロマチンの相互作用の検討を可能にし、クロマチンの相互作用に特異性を追加します。

<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> 4から20パーセント勾配TBEゲル</td><td>インビトロジェン</td><td> EC6225BOX </td><td>ステップB、6.3 </td></tr><tr><td> PEGを用いた5×T4 DNAリガーゼバッファー</td><td>インビトロジェン</td><td> 46300018 </td><td>ステップB、2.2 </td></tr><tr><td> 6パーセントTBEゲル</td><td>インビトロジェン</td><td> EC6263BOX </td><td>ステップB、6.8 </td></tr><tr><td> AgilentのDNA 100アッセイ</td><td>アジレント·テクノロジー</td><td> 5067-1504 </td><td rowspan="3">ステップB、7.1 </td></tr><tr><td> Agilentの高感度DNAアッセイ</td><td>アジレント·テクノロジー</td><td> 5067-4626 </td></tr><tr><td>アジレントバイオアナライザ2100 </td><td>アジレント·テクノロジー</td><td> G2940CA </td></tr><tr><td> （スピン-X）管フィルタを遠心</td><td>コー​​ニング&lt;/ TD&gt; <td> CLS8160 </td><td>ステップB、3.7と6.9 </td></tr><tr><td>ダークリーダーネーター</td><td>クレア化学研究所</td><td> DR46B </td><td>ステップB、6.8 </td></tr><tr><td>デジタル超音波処理細胞攪乱</td><td>ブランソン</td><td> 450D-0101063591 </td><td> 、4.3ステップ</td></tr><tr><td> DynaMag-2磁石（磁性粒子コンセントレータ） </td><td>インビトロジェン</td><td> 123-21D </td><td>洗浄工程のすべての磁気ビーズのために、7.5.1ステップBステップ</td></tr><tr><td> DynaMag-15マグネット（磁気粒子コンセントレータ） </td><td>インビトロジェン</td><td> 123.01D </td><td> 、5とステップ6 </td></tr><tr><td> DynaMag-PCR（磁性粒子コンセントレータ） </td><td>インビトロジェン</td><td> 49から2025 </td><td>ステップB、6.5 </td></tr><tr><td> 大腸菌DNAポリメラーゼI </td><td> NEB </td><td> M0209 </td><td>ステップB、5.6 </td></tr><tr><td> GlycoBlue </td><td> Ambionの</td><td> AM9516 </TD&gt; <td> 、7.5.1ステップB、4.3および6.5のステップ</td></tr><tr><td>イルミナCBOTクラスタ生成システム</td><td>イルミナ</td><td> SY-301-2002 </td><td>ステップB、7.4 </td></tr><tr><td>イルミナGenome AnalyzerのIIxは</td><td>イルミナ</td><td> SY-301-1301 </td><td>ステップB、7.5 </td></tr><tr><td>イルミナPEプライマー</td><td>イルミナ</td><td> PE-102-1004 </td><td>ステップB、5.4（必要に応じて） </td></tr><tr><td>インテリミキサー</td><td> Palicoバイオ</td><td> RM-2L </td><td>ステップB、回転を持つ任意のインキュベーション</td></tr><tr><td>ライトサイクラー480リアルタイムPCRシステム</td><td>ロシュ社</td><td> 04 640 268 001 </td><td> 、7.5.3ステップBステップ、7.3 </td></tr><tr><td> LightCycler480 DNAのSYBR Green Iマスターミックス</td><td>ロシュ社</td><td> 03 752 186 001 </td><td>ステップB、7.5.3 </td></tr><tr><td> M-280ストレプトアビジンDynabeads®を</td><td>インビトロジェン</td><td> 11206D </td><td>ステップB、5.2 </td></tr> &lt;TR&gt; <td>磁気（ダイナ）プロテインG </td><td>インビトロジェン</td><td> 100.03D </td><td> 、5.1.1および5.2.1のステップ</td></tr><tr><td>高密度（2ミリリットル）をMaXtract </td><td>キアゲン</td><td> 129056 </td><td> 、7.5.1ステップ</td></tr><tr><td>高密度（50ミリリットル）をMaXtract </td><td>キアゲン</td><td> 129073 </td><td>ステップB、4.2 </td></tr><tr><td> MmeI </td><td> NEB </td><td> R0637 </td><td> R0637 </td></tr><tr><td>アーゼONEリボヌクレアーゼ</td><td>プロメガ</td><td> M426C </td><td>ステップB、4.8 </td></tr><tr><td> RNAポリメラーゼII（8WG16）モノクローナル抗体</td><td> Covance </td><td> MMS-126R </td><td>ステップ5.2.4 </td></tr><tr><td>オークリッジ遠心管（ポリプロピレン） </td><td>ナルゲン</td><td> 3119-0050 </td><td>ステップ3.1 </td></tr><tr><td>オークリッジ遠心チューブ（テフロンFEP） </td><td>ナルゲン</td><td> 3114-0050 </td><td>ステップB、4.3 </td></tr><tr><td> Phusイオンハイ·フィデリティマスターミックス</td><td> Finnzymes </td><td> F-531 </td><td> Bは、ステップ6に</td></tr><tr><td>ピコグリーン（クワント-IT）のdsDNA試薬</td><td>インビトロジェン</td><td> P11495 </td><td>ステップ7.5.2 </td></tr><tr><td>ポリスチレン丸底試験管</td><td> BDバイオサイエンス</td><td> 352057 </td><td> 4.1ステップ</td></tr><tr><td>プロテアーゼインヒビターカクテル錠（完全、EDTAフリー） </td><td>ロシュ社</td><td> 11873580001 </td><td> 、1.6以降のステップ</td></tr><tr><td>プロテイナーゼK溶液（20mg/ml） </td><td> Fermentas </td><td> E00491 </td><td> 、4.4、7.5.1ステップ ステップB 4.1 </td></tr><tr><td> T4 DNAリガーゼ</td><td> Fermentas </td><td> EL0013 </td><td>ステップB、2.2、3.9および5.4 </td></tr><tr><td> T4 DNAリガーゼ緩衝液（NEB） </td><td> NEB </td><td> B0202S </td><td>ステップB、3.3および3.9 </td></tr><tr><td> T4 DNAポリメラーゼ</td><td>プロメガ</td><td> M4215 </td><td>ステップB、1.2 </td></tr><tr><td> T4 DNAポリヌクレオチドキナーゼ</td><td> NEB </td><td> M0201 </td><td>ステップB、3.3 </td></tr><tr><td> TruSeq SBSキットV5-GA </td><td>イルミナ</td><td> FC-104-5001 </td><td>イルミナGenome AnalyzerのIIxは、システムのリージェンツ</td></tr><tr><td> TruSeq PEクラスタキットV2-CBOT-GA </td><td>イルミナ</td><td> PE-300-2001 </td><td>イルミナCBOTクラスタ発電システムで使用できるようにするには</td></tr><tr><td> SYBR Green Iを</td><td>インビトロジェン</td><td> S-7585 </td><td>ステップB、6.3および6.8 </td></tr><tr><td> X細胞SureLockミニ細胞電気泳動システム</td><td>インビトロジェン</td><td> EI0001 </td><td>ステップB、6.3および6.8 </td></tr></tbody></table><table border="1"><tbody><tr><td> 名 </td><td colspan="2"> シーケンス </td><td> コメント </td></tr><tr><td rowspan="2">ビオチン化したハーフリンカーA（200ng/μl） </td><td>トップ</td><td> 5 &#39;GG CCG CGA / iBiodT / ATC TTA TCC AACの3&#39; </td><td> 250ナノモル規模HPLC精製された内部ビオチンDT（9） </td></tr><tr><td>ボット</td><td> 5 &#39;GTT GGA TAA GAT ATC GC 3&#39; </td><td> 250ナノモルスケールHPLC精製</td></tr><tr><td rowspan="2">ビオチン化したハーフリンカーB（200ng/μl） </td><td>トップ</td><td> 5 &#39;GGC CGC GA / iBiodT / ATA CAT TCC AACの3&#39; </td><td> 250ナノモル規模HPLC精製された内部ビオチンDT（9） </td></tr><tr><td>ボット</td><td> 5 &#39;GTT GGA ATG TAT ATC GC 3&#39; </td><td> 250ナノモルスケールHPLC精製</td></tr><tr><td rowspan="2">非ビオチン化したハーフリンカー（200ng/μl） </td><td>トップ</td><td> 5 &#39;CGC GGC GAT ATC GGA TCC AACの3&#39; </td><td> 250ナノモル規模PCRグレード</td></tr><tr><td>ボット</td><td> 5 &#39;GTT GGA TCC GAT ATC GC 3&#39; </td><td> 250ナノモル規模PCRグレード</td></tr><tr><td rowspan="2"> GS20アダプター（200ng/μl） </td><td>トップ</td><td> 5 &#39;CCA TCT CAT CCC TGC GTG TCC CATCTG TTC CCT CCC TGT CTC AGN N 3 &#39; </td><td> 250ナノモル規模PCRグレード</td></tr><tr><td>ボット</td><td> 5&#39;CTG AGA CAG GGA GGG AAC AGA TGG GAC ACG CAG GGA TGA GAT GG 3 &#39; </td><td> 250ナノモル規模PCRグレード</td></tr><tr><td rowspan="2"> GS20アダプターB（200ng/μl） </td><td>トップ</td><td> 5 &#39;CTG C​​AC AGA GCA ACA GGG GAT AGG CAA GGC ACA CAG GGG ATA GG 3&#39; </td><td> 250ナノモル規模PCRグレード</td></tr><tr><td>ボット</td><td> 5 &#39;CCT ATC CCC TGT GTG CCT TGC CTA TCC CCT GTT TGT GCG CTC AGN N 3&#39; </td><td> 250ナノモル規模PCRグレード</td></tr><tr><td rowspan="2">イルミナ-NNアダプタ</td><td>トップ</td><td> 5 &#39;ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TC 3&#39; </td><td> 250ナノモルスケールHPLC精製は、ステップB、5.4を参照してください。 </td></tr><tr><td>ボット</td><td> 5 &#39;リン - GAT CGG AAG AGC TCA GGT GCA GGA ATG CCG AG 3&#39; </td><td> 250ナノモルスケールHPLC精製は、5 &#39;末端は、ステップBを参照してください5.4リン酸化</td></tr><tr><td>イルミナ1から454（フォワード）プライマー（10μM） </td><td colspan="2"> 5 &#39;AAT GAT ACG GCG ACC ACC GAG ATC TAC ACC CTA TCC CCT GTG TGC CTT G 3&#39; </td><td> 250ナノモル規模PCRグレード</td></tr><tr><td>イルミナ2から454（リバース）プライマー（10μM） </td><td colspan="2"> 5 &#39;CAA GCA GAA GAC GGC ATA CGA CGG GAT TCC ATC TCA TCC CTG C​​GT GTCの3&#39; </td><td> 250ナノモル規模PCRグレード</td></tr><tr><td>定量PCRプライマー1.1（10μM） </td><td colspan="2"> 5 &#39;→3 AT AAT GAT ACG GCG ACC ACC GAG&#39; </td><td> 10nmole規模PCRグレード</td></tr><tr><td>定量PCRプライマー2.1（10μM） </td><td colspan="2"> 5 &#39;CAA GCA GAA GAC GGC ATA CGA 3&#39; </td><td> 10nmole規模PCRグレード</td></tr><tr><td>イルミナの3から454シークエンシングプライマー（100μM） </td><td colspan="2"> 5&#39;TGC GTG TCC CAT TTC CTG C​​CT CCC TGT CTC AG 3 &#39; </td><td> 100nmoleスケールHPLC精製</td></tr><tr><td>イルミナの4から454の配列決定プライマー（100μM） </td><td colspan="2"> 5&#39;GTG CCT TGC CTA TCC CCT GTT TGT GCG CTC AG 3 &#39; </td><td> 100nmole規模HPLC精製</td></tr></tbody></table>統合されたDNAテクノロジー（IDT）からオリゴおよびアダプタのテーブル。注文オリゴは、10前述と同様にリンカーおよびアダプタを準備します。ハーフリンカーとアダプタあらかじめ準備され、-20℃で数ヶ月の​​保存が可能

chromatin interaction analysis with paired-end tag sequencing (chia-pet) for mapping chromatin interactions and understanding transcription regulation

ゲノムは、ミクロンサイズの核のスペース内に高次のコンフォメーション7、2、12を採用し 、三次元構造に編成されています。このようなアーキテクチャは、ランダムではなく、遺伝子のプロモーターおよび調節エレメント13との間の相互作用を伴う。特定の調節配列に転写因子の結合は、転写調節と協調1、14のネットワークをもたらす。 ペアエンドタグシーケンシング（CHIA-PET）によるクロマチンの相互作用の解析は、これらの高次クロマチン構造の5,6を識別するために開発されました。セルが固定されており、相互作用する遺伝子座は、共有結合、DNA-タンパク質のクロスリンクによってキャプチャされます。非固有のノイズを最小限に抑え、複雑さを軽減するだけでなく、クロマチンの相互作用分析の特異性を向上させるために、クロマチン免疫沈降（ChIP）は、近接ライゲーションの前に興味のあるクロマチンの断片を豊かにするために、特定のタンパク質因子に対して使用されている。ハーフリンカーを含むライゲーションは、その後、個々のクロマチン複合体の中で一緒につながれたDNA断片のペアの間に共有リンクを形成します。ハーフリンカーに隣接MmeI制限酵素サイトはMmeI消化時のペアの終了タグ - リンカー - タグ構造（PETS）の抽出を可能にします。ハーフリンカーはビオチン化されているように、これらのPETの構造は、ストレプトアビジン磁気ビーズを用いて精製されています。精製したPETSは、次世代シーケンシング·アダプタとの相互作用フラグメントのカタログのようなイルミナGenome Analyzerなどの次世代シーケンサーを経由して生成されますと連結されています。マッピングおよびバイオインフォマティクス解析は、チップに富む結合部位とチップ濃縮クロマチンの相互作用8を識別するために実行されます。 我々は、チップの品質がCHIA-PETライブラリの結果に大きな役割を果たしている特にCHIA-PETプロトコルは、チップの準備の重要な側面を示すためにビデオを制作しています。プロトコルがあるよう非常に長い、唯一の重要なステップは、ビデオで表示されます。

Watch this Scientific Journal Video about Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation at JoVE.c

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

ペアエンドタグシーケンシング（CHIA-PET）によるクロマチンの相互作用分析のための方法です。<em&gt; de novoの</em転写制御の理解を深めるためのクロマチンの相互作用&gt;検出。

クロマチンの相互作用のマッピングと転写調節を理解するためのペアエンドタグシーケンシング（CHIA-PET）によるクロマチンの相互作用の解析

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

chromatin-interaction-analysis-with-paired-end-tag-sequencing-chia

Research

JoVE Journal

Biology

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

30.5K ビュー.  Agency for Science, Technology and Research, Singapore. ペアエンドタグシーケンシング（CHIA-PET）によるクロマチンの相互作用分析のための方法です。 de novoの検出。

ビデオ: Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

人間の脳組織のためのクロマチンアッセイ

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

生物学

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

10ナノグラムのトータルRNAからシングルリードとペアエンドのmRNA - seqのイルミナライブラリ

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

を使用してクロマチン免疫沈降（ChIP）ショウジョウバエ組織

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA精製によってクロマチンの単離（チャープ）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

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 ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

ホールマウント胚珠内でのクロマチン修飾や核構造の定量的、単一細胞分析のための効率的な方法<em&gt;シロイヌナズナ</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

発生と人間INO80クロマチンリモデリング複合体とSubcomplexesの精製

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

シロイヌナズナタンパク質-DNA相互作用の同定のための免疫沈降アッセイクロマチン<em&gt;インビボ</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

enChIPによって特定のゲノム領域と関連する分子の同定の単離

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

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).