この作業は研究とウィスコンシン財団の大学Morgridge研究所からの資金によって支えられている。我々は編集支援のためクリスタイーストマンに感謝。

<ol>
	<li>Tang, F., Barbacioru, C., Wang, Y., Nordman, E., Lee, C., Xu, N., Wang, X., Bodeau, J., Tuch, B. B., Siddiqui, A., Lao, K., Surani, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA-Seq+whole-transcriptome+analysis+of+a+single+cell.">mRNA-Seq whole-transcriptome analysis of a single cell.</a> Nature Methods. 6, 377-382 (2009).</li><li>Armour, C. D., Castle, J. C., Chen, R., Babak, T., Loerch, P., Jackson, S., Shah, J. K., Dey, J., Rohl, C. A., Johnson, J. M., Raymond, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+transcriptome+profiling+using+selective+hexamer+priming+for+cDNA+synthesis.">Digital transcriptome profiling using selective hexamer priming for cDNA synthesis.</a> Nature Methods. 6, 647-649 (2009).</li><li>Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+quantifying+mammalian+transcriptomes+by+RNA-Seq.">Mapping and quantifying mammalian transcriptomes by RNA-Seq.</a> Nature Methods. 5, 621-628 (2008).</li><li>Mamanova, L., Andrews, R. M., James, K. D., Sheridan, E. M., Ellis, P. D., Langford, C. F., Ost, T. W., Collins, J. E., Turner, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRT-seq+amplification-free,+strand-specific+transcriptome+sequencing.">FRT-seq amplification-free, strand-specific transcriptome sequencing.</a> Nature Methods. 5, 130-132 (2010).</li><li>Sengupta, S., Ruotti, V., Bolin, J., Elwell, A., Hernandez, A., Thomson, J., Stewart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+consistent,+fully+representative+mRNA-Seq+libraries+from+ten+nanograms+of+total+RNA.">Highly consistent, fully representative mRNA-Seq libraries from ten nanograms of total RNA.</a> Biotechniques. 49, 898-904 (2010).</li><li>Langmead, B., Trapnell, C., Pop, M., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+and+memory-efficient+alignment+of+short+DNA+sequences+to+the+human+genome.">Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.</a> Genome. Biology. 10, R25-R25 (2009).</li><li>Li, B., Ruotti, V., Stewart, R. M., Thomson, J. A., Dewey, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq+gene+expression+estimation+with+read+mapping+uncertainty.">RNA-Seq gene expression estimation with read mapping uncertainty.</a> Biotechniques. 26, 493-500 (2010).</li><li>Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., Jones, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li></ol>

著者らは、JATが携帯電話ダイナミクス国際（CDI）の創設者、stockowner、コンサルタントおよびボードメンバーであることを開示している。彼はまたの科学顧問としての役割を果たし、タクティクスII幹細胞のベンチャーズの経済的利益を持っています。

ペアエンドライブラリを作るための現在のプロトコルは、1μgの9〜2.5μgのトータルRNAの10開始量の間に必要とする。ここでは、このメソッドは許可されている両方の単一の読み取りとペアエンドイルミナシーケンシングライブラリとショーを準備する私たちのリニアT7増幅系（T7LA）メソッドを提示するのに匹敵するデータを生成するトータルRNAのような低10 ngのからのライブラリの生成1000倍以上の出発原料（10μgのトータルRNA）から作られた最小限の増幅（MinAmp）ライブラリ。 10 ngのライブラリーは、遺伝子の類似の総数を特定するものではなく、また（図3aとb）類似している遺伝子発現の署名を生成しません。また、T7LA法により製造された単一の読み取りとペアエンドライブラリの両方は、研究者がどちらのプロトコルで作られたライブラリによって生成されたデータを比較できるようになる、（図3c）は互いに非常によく似ています。これらのライブラリは、ヒト胚性幹細胞のRNAから調製されて以来、我々が検索ライブラリのうち、30幹細胞特異的遺伝子のため、ほぼこれらの遺伝子のすべて（93から100パーセント）はこのように我々のプロトコルを検証する、トータルRNAを（図4）出発の少なくとも10 ngのから作られたライブラリによって識別されていることがわかります。我々は、我々のプロトコルは、特にそのようなフローソートされた細胞または出発物質が制限され、レーザーマイクロ解剖組織としての状況では、研究者にとって非常に有用ではないかと思います。このような状況では、我々のプロトコルは、我々のプロトコルが開始RNAの大きさの少なくとも3桁でほぼ同等の発現プロファイルを生成するので、はるかに大きいの開始量から作られたライブラリに匹敵する遺伝子発現データの生成をできるようになります。

<table border="1"><tbody><tr><td>会社</td><td>キット/試薬</td><td>カタログ＃ </td><td>特別なコメント</td></tr><tr><td>アンビオン</td><td>断片化試薬</td><td> AM8740 </td><td></td></tr><tr><td>アンビオン</td><td>ライナーのアクリルアミド</td><td> AM9520 </td><td></td></tr><tr><td>アンビオン</td><td> MEGAscript T7キット</td><td> AM1334 </td><td></td></tr><tr><td>アンビオン</td><td>非DEPCは、ヌクレアーゼフリーの水を扱わ</td><td> AM9932 </td><td></td></tr><tr><td> Fermentas </td><td> dNTPのセット</td><td> R0181 </td><td></td></tr><tr><td> Fermentas </td><td>メチル化のdNTP </td><td> R0431 </td><td>唯一のシングルリードのサンプル調製用</td></tr><tr><td>イルミナ</td><td> TruSeq SRクラスタ生成キットV5 </td><td> GD - 203 - 5001 </td><td>唯一のシングルリードのサンプル調製用</td></tr><tr><td>イルミナ</td><td> TruSeqのSeqキットV5 36サイクル</td><td> FC - 104 - 5001 </td><td></td></tr><tr><td>イルミナ</td><td>チップのSeqサンプル調製キット</td><td> IP - 102 - 1001 </td&gt; <td>シングルリードサンプル調製用のみ。 1 CAT＃E6040S設定NEB DNAサンプルプレップマスターミックスに置き換えることができます。 </td></tr><tr><td>イルミナ</td><td> TruSeq PEクラスタ生成キットV5 </td><td> PE - 203 - 5001 </td><td>ペアエンドサンプル調製用のみ</td></tr><tr><td>イルミナ</td><td>ペアエンドサンプル前処理キッ​​ト</td><td> PE - 102 - 1001 </td><td>ペアエンドサンプル調製用のみ</td></tr><tr><td>インビトロジェン</td><td> 1キロバイトプラスはしご</td><td> 10787-018 </td><td></td></tr><tr><td>インビトロジェン</td><td>大腸菌のRNase H </td><td> 18021-014 </td><td></td></tr><tr><td>インビトロジェン</td><td> RNaseのアウト</td><td> 10777-019 </td><td></td></tr><tr><td>インビトロジェン</td><td>第二鎖バッファー5倍</td><td> 10812-014 </td><td></td></tr><tr><td>インビトロジェン</td><td>大腸菌のDNAポリメラーゼ</td><td> 18010-017 </td><td></td></tr><tr><td>インビトロジェン</td><td> E -ゲルSYBR安全な2パーセント</td><td> G521802 </td><td></td></tr><tr><td>インビトロジェン</td><td>上付きIII（5X FSバッファおよび0.1M DTT付き） </td><td> 18080-085 </td><td></td></tr><tr><td>インビトロジェン</td><td>トリゾール</td><td> 12183555 </td><td></td></tr><tr><td>インビトロジェン</td><td> RNA quibitアッセイキット</td><td> Q32852 </td><td></td></tr><tr><td>インビトロジェン</td><td> dsDNAをHS量子ビットアッセイキット</td><td> Q32851 </td><td></td></tr><tr><td>インビトロジェン</td><td>大腸菌DNAリガーゼ</td><td> 18052-019 </td><td></td></tr><tr><td>インビトロジェン</td><td> T4 DNAポリメラーゼ</td><td> 18005-025 </td><td></td></tr><tr><td>インビトロジェン</td><td>超高純度のDNase RNaseの水</td><td> 10977-015 </td><td></td></tr><tr><td>インビトロジェン</td><td>スーパースクリプトII二本鎖cDNA合成キット</td><td> 11917-020 </td><td></td></tr><tr><td>インビトロジェン</td><td>ランダムプライマー（Invitrogen社） </td><td> 48190-011 </td><td>ペアエンドサンプル調製用のみ</td></tr><tr><td> IDT </td><td> Not1Nonamer Bのプライマー</td><td> N / </td><td>シングルリードSAM用PLE準備のみ</td></tr><tr><td> IDT </td><td>オリゴdT T7 </td><td> N / </td><td></td></tr><tr><td> NEB </td><td> NOT1消化キット</td><td> R0189S </td><td>唯一のシングルリードのサンプル調製用</td></tr><tr><td>キアゲン</td><td>をRNeasyアプライキット</td><td> 74204 </td><td></td></tr><tr><td>キアゲン</td><td> RNeasyミニキット（50） </td><td> 74104 </td><td></td></tr><tr><td>キアゲン</td><td>ゲル精製キット</td><td> 28604 </td><td></td></tr><tr><td>キアゲン</td><td> DNase処理セット</td><td> 79254 </td><td></td></tr><tr><td>キアゲン</td><td>したRNeasyアプライキット</td><td> 74204 </td><td></td></tr><tr><td> Zymo研究</td><td> DNAクリーンとコンセントレータ（250X） </td><td> D4014 </td><td></td></tr></tbody></table>

single read and paired end mrna-seq illumina libraries from 10 nanograms total rna

mRNAのseqで全トランスクリプトームの配列決定は、現在、グローバルな遺伝子発現、突然変異、対立遺伝子特異的発現および他のゲノムワイドな解析を実行するために広く使用されている。 mRNAのSeqはさらに、非塩基配列ゲノムの遺伝子発現解析のためのゲートを開きます。 mRNAのSeqは高感度、広いダイナミックレンジを提供し、サンプル中の転写産物のコピー数を測定することができます。イルミナ社のGenome Analyzerは、単一の長いリードを特徴とする&quot;ペアエンド&quot;のアプローチは、両方の両端に配列決定されている。大比較的短いシーケンスの数（&gt; 10 7）（&lt;150 bp）の読み取りの配列決定を行い、選択的スプライシングのジャンクションを追跡することができます、挿 ​​入や削除、およびde novoのトランスクリプトームアセンブリするのに便利です。 研究者が直面する大きな課題の一つは、出発原料の限られた量です。トータルRNA mを開始例えば、細胞がレーザーマイクロダイセクションで採取される実験では、利用可能なナノグラムの措置をAYがつきます。このようなサンプルからのmRNA - seqのライブラリーの調製は、1、2を説明するが、バイアスを導入することが重要なPCR増幅を伴うしている。最小限のPCR増幅を持つ他のRNA - seqのライブラリー構築の手順は3、4を発表したが、全体のRNAを開始するのマイクログラムの量を必要とされている。 ここでは重要なPCR増幅を回避し、トータルRNAのわずか10ナノグラムを必要とするライブラリの準備のためのmRNA - seqのシーケンスのためのイルミナGenome AnalyzerのIIのプラットフォーム用のプロトコルについて説明します。このプロトコルは、それが重要な増幅バイアスを導入せず、方向ライブラリーを作製することが示されたシングルエンドシーケンシング5、の説明で述べたと検証してきたが、ここでは、ペアエンドのプロトコルとして使用するためにさらにそれを検証する。我々は選択的に&quot;T7造語、T7ベースのEberwineリニア増幅法を用いてトータルRNAを起動ポリアデニルメッセンジャーRNAを増幅LA&quot;（T7リニア増幅）。mRNAの断片化されて増幅されたポリ、最終的なシーケンスのライブラリを生成するライゲーション転写し、アダプタを逆にする。双方単一の読み取りとペアエンドの実行では、配列がヒトのトランスクリプトーム6にマップされているとなるように正規化複数の実行からのデータを比較することができます。我々は試料7を比較するときRPKMに優れた尺度である百万ごとの転写産物の単位で遺伝子発現の測定（TPM）を、報告する。

Watch this Scientific Journal Video about Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA at JoVE.com

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

ここでは、T7リニアRNA増幅に基づく遺伝子発現解析用のライブラリーのシークエンシングの両方シングルリードとペアエンドイルミナのmRNA - seqの製造方法を説明します。このプロトコルは、トータルRNAを始めるのは10ナノグラムを必要とし、全体の転写産物を表す非常に一貫性のあるライブラリを生成します。

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 ...

single-read-paired-end-mrna-seq-illumina-libraries-from-10-nanograms

Research

JoVE Journal

Biology

39.0K ビュー.  Morgridge Institute for Research. ここでは、T7リニアRNA増幅に基づく遺伝子発現解析用のライブラリーのシークエンシングの両方シングルリードとペアエンドイルミナのmRNA - seqの製造方法を説明します。このプロトコルは、トータルRNAを始めるのは10ナノグラムを必要とし、全体の転写産物を表す非常に一貫性のあるライブラリを生成します。

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

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

メタゲノムライブラリの大規模スクリーン

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

生物学

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

Bio - Rad社のTotal RNA Kitを使​​用してNeuroprecursor細胞からのRNA抽出

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

人間の死後の脳組織における単一細胞から、高品質のRNAを取得

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

メタゲノムライブラリからセルラーゼ活性をBiominingためのハイスループットスクリーニング

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HeLa細胞のトランスフェクションに続いてGaussiaルシフェラーゼmRNA のインビトロ転写とキャッピングの

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

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

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

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

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

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 ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: 細胞型特異エピゲノムのプロファイリング

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

単核RNAシーケンシングのための成体マウス腎臓からの核分離

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

凍結哺乳類組織からの単一核の単離とプロファイリング

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

著者スポットライト:創薬の強化 - 凍結組織からの核抽出のための自動化され標準化されたプロトコルの開発 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.