Este trabalho foi financiado por fundos do Instituto Morgridge de Pesquisas e da Universidade de Wisconsin Foundation. Agradecemos a Krista Eastman para a assistência editorial.

<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>

Os autores revelam que JAT é um dos fundadores, stockowner, consultor e membro do conselho da Cellular Dynamics International (CDI). Ele também atua como assessor científico e tem interesses financeiros em Tactics II Ventures células-tronco.

Protocolos atuais para fazer bibliotecas final emparelhados exigem entre 1 mg 9-2,5 mg 10 montante inicial de RNA total. Aqui apresentamos a nossa linear de amplificação T7 método (T7LA) com base para preparar a única bibliotecas final ler e emparelhado Illumina seqüenciamento e mostrar que este método permite a geração de bibliotecas de tão baixo quanto 10 ng de RNA total, produzindo dados que é comparável à de minimamente amplificado (MinAmp) bibliotecas feitas a partir de 1000 o material de partida vezes mais (10 mg RNA total). As 10 bibliotecas ng não só identificar semelhante o número total de genes, mas também produzem assinaturas de expressão gênica que são semelhantes (Figuras 3a e b). Além disso, tanto o single bibliotecas final ler e pares produzidas pelo método T7LA são muito semelhantes entre si (Figura 3c), que permite aos pesquisadores comparar os dados gerados pelas bibliotecas feitas a partir de qualquer protocolo. Uma vez que essas bibliotecas foram preparados a partir de RNA humano de células-tronco embrionárias, buscamosde 30 genes de células-tronco específicas entre as bibliotecas e descobrir que quase todos esses genes (93-100%) são identificadas pelas bibliotecas feita a partir de pelo menos 10 ng de RNA total de partida (Figura 4), validando o nosso protocolo. Acreditamos que o nosso protocolo seria muito útil para pesquisadores, especialmente em circunstâncias como as células de fluxo classificados ou a laser micro-dissecados tecidos onde o material de partida é limitante. Em tais circunstâncias, o nosso protocolo permitiria a geração de dados de expressão gênica comparável às bibliotecas feita a partir de quantidades muito maiores de partida desde o nosso protocolo produz perfis de expressão comparáveis ​​entre pelo menos 3 ordens de magnitude de RNA de partida.

<table border="1"><tbody><tr><td> Companhia </td><td> Kit / Reagente </td><td> Catalogo </td><td> Comentários especiais </td></tr><tr><td> Ambion </td><td> O reagente de fragmentação </td><td> AM8740 </td><td></td></tr><tr><td> Ambion </td><td> A acrilamida Liner </td><td> AM9520 </td><td></td></tr><tr><td> Ambion </td><td> MEGAscript T7 Kit </td><td> AM1334 </td><td></td></tr><tr><td> Ambion </td><td> DEPC não tratados água livre de nuclease </td><td> AM9932 </td><td></td></tr><tr><td> Fermentas </td><td> dNTP set </td><td> R0181 </td><td></td></tr><tr><td> Fermentas </td><td> dNTPs metilado </td><td> R0431 </td><td> Para preparação de amostras Único Somente leitura </td></tr><tr><td> Illumina </td><td> TruSeq SR Cluster Generation kit v5 </td><td> GD-203-5001 </td><td> Para preparação de amostras Único Somente leitura </td></tr><tr><td> Illumina </td><td> TruSeq Seq Kit v5 36 ciclos </td><td> FC-104-5001 </td><td></td></tr><tr><td> Illumina </td><td> Chip Amostra Seq Prep Kit </td><td> IP-102-1001 </td&gt; <td> Para preparação de amostras Único Somente leitura. Pode ser substituído por amostra NEB prep DNA Mix Master Set 1 Cat # E6040S </td></tr><tr><td> Illumina </td><td> TruSeq PE Cluster Generation kit v5 </td><td> PE-203-5001 </td><td> Para prep emparelhado final amostra apenas </td></tr><tr><td> Illumina </td><td> Emparelhado End Sample Prep Kit </td><td> PE-102-1001 </td><td> Para prep emparelhado final amostra apenas </td></tr><tr><td> Invitrogen </td><td> 1kb ladder mais </td><td> 10787-018 </td><td></td></tr><tr><td> Invitrogen </td><td> E. coli Rnase H </td><td> 18021-014 </td><td></td></tr><tr><td> Invitrogen </td><td> Rnase Out </td><td> 10777-019 </td><td></td></tr><tr><td> Invitrogen </td><td> Tampão vertente 2 5x </td><td> 10812-014 </td><td></td></tr><tr><td> Invitrogen </td><td> E. coli DNA polimerase </td><td> 18010-017 </td><td></td></tr><tr><td> Invitrogen </td><td> E-gel SYBR 2% seguro </td><td> G521802 </td><td></td></tr><tr><td> Invitrogen </td><td> Sobrescrito III (com 5X FS buffer e DTT 0,1 M) </td><td> 18080-085 </td><td></td></tr><tr><td> Invitrogen </td><td> Trizol </td><td> 12183555 </td><td></td></tr><tr><td> Invitrogen </td><td> RNA ensaio quibit kit </td><td> Q32852 </td><td></td></tr><tr><td> Invitrogen </td><td> dsDNA HS qubit ensaio kit </td><td> Q32851 </td><td></td></tr><tr><td> Invitrogen </td><td> E. coli DNA ligase </td><td> 18052-019 </td><td></td></tr><tr><td> Invitrogen </td><td> Polimerase DNA T4 </td><td> 18005-025 </td><td></td></tr><tr><td> Invitrogen </td><td> Ultra Pure Dnase água Rnase </td><td> 10977-015 </td><td></td></tr><tr><td> Invitrogen </td><td> Sobrescrito II dupla kit síntese de cDNA encalhado </td><td> 11917-020 </td><td></td></tr><tr><td> Invitrogen </td><td> Primer aleatório (Invitrogen) </td><td> 48190-011 </td><td> Para prep emparelhado final amostra apenas </td></tr><tr><td> IDT </td><td> Not1Nonamer cartilha B </td><td> N / A </td><td> Para Single Leia samsoas prep apenas </td></tr><tr><td> IDT </td><td> Oligo dT T7 </td><td> N / A </td><td></td></tr><tr><td> NEB </td><td> Not1 kit digestão </td><td> R0189S </td><td> Para preparação de amostras Único Somente leitura </td></tr><tr><td> Qiagen </td><td> Rneasy Minelute kit </td><td> 74204 </td><td></td></tr><tr><td> Qiagen </td><td> RNEasy Mini Kit (50) </td><td> 74104 </td><td></td></tr><tr><td> Qiagen </td><td> Kit de purificação de gel </td><td> 28604 </td><td></td></tr><tr><td> Qiagen </td><td> DNase set </td><td> 79254 </td><td></td></tr><tr><td> Qiagen </td><td> Rneasy MinElute kit </td><td> 74204 </td><td></td></tr><tr><td> Zymo Research </td><td> DNA limpo e concentrador (250X) </td><td> D4014 </td><td></td></tr></tbody></table>

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

Seqüenciamento do transcriptoma inteiro por mRNA Seq-se amplamente utilizado para realizar a expressão gênica global, mutação, expressão alelo-específico e outras análises genômicas. mRNA-Seq ainda abre o portão para a análise de expressão gênica de genomas seqüenciados não. mRNA Seq oferece alta sensibilidade, uma grande gama dinâmica e permite a medição do número de cópias transcrição em uma amostra. Analisador Illumina genoma realiza sequenciamento de um grande número (&gt; 10 7) da seqüência relativamente curta lê (&lt;150 pb). O &quot;emparelhado fim&quot; abordagem, em que uma leitura única e longa é seqüenciado em ambas as extremidades a sua, permite o rastreamento junções splice alternativo , inserções e deleções, e é útil para a montagem de transcriptoma novo. Um dos grandes desafios enfrentados pelos pesquisadores é uma quantidade limitada de matéria-prima. Por exemplo, em experimentos onde as células são colhidas por laser micro-dissecção, disponível a partir de RNA total de may medida em nanogramas. Preparação de mRNA Seq-bibliotecas a partir de tais amostras foram descritas 1, 2, mas envolve a amplificação por PCR significativo que pode introduzir viés. Outros RNA-Seq procedimentos de construção da biblioteca com a amplificação PCR mínima foram publicados 3, 4, mas requerem quantidades micrograma de RNA total de partida. Aqui nós descrevemos um protocolo para a plataforma Illumina Genome Analyzer II para mRNA Seq-seqüenciamento para a preparação de biblioteca que evita a amplificação PCR significativa e requer apenas 10 nanogramas de RNA total. Embora este protocolo foi descrito anteriormente e validado para um único fim de seqüenciamento 5, onde foi mostrado para produzir bibliotecas direcional sem introduzir viés de amplificação significativa, aqui nós validá-lo para utilização como um protocolo final emparelhados. Nós seletivamente amplificam RNA mensageiro polyadenylated de iniciar RNA total usando o T7 com base Eberwine método de amplificação linear, cunhado &quot;T7LA &quot;(T7 amplificação linear). Amplificada O poli-A mRNAs são fragmentados, reverso transcrito e adaptador ligado para produzir a biblioteca seqüenciamento final. Para ambos única corre final ler e emparelhado, as seqüências são mapeados para o transcriptoma humano 6 e normalizados para que dados de várias execuções podem ser comparados. Relatamos a medida de expressão gênica em unidades de transcrições por milhão (TPM), que é uma medida superior a RPKM quando se comparam amostras 7.

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

Aqui nós descrevemos um método para a preparação de ambos única leitura e emparelhado final Illumina mRNA Seq-libraries seqüenciamento para análise de expressão gênica com base na amplificação T7 RNA linear. Este protocolo requer apenas 10 nanogramas de começar RNA total e gera bibliotecas altamente consistentes representando transcrições todo.

Leia única e emparelhados End mRNA-Seq Bibliotecas Illumina de 10 nanogramas RNA total

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 visualizações.  Morgridge Institute for Research. Aqui nós descrevemos um método para a preparação de ambos única leitura e emparelhado final Illumina mRNA Seq-libraries seqüenciamento para análise de expressão gênica com base na amplificação T7 RNA linear. Este protocolo requer apenas 10 nanogramas de começar RNA total e gera bibliotecas altamente consistentes representando transcrições todo.

Vídeo: 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.

Grande Escala Telas de Bibliotecas metagenomic

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

Biologia

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

Extração de RNA a partir de células Neuroprecursor Usando o Bio-Rad Total de RNA Kit

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.

Obtenção de RNA de alta qualidade a partir de populações de células no tecido humano único post-mortem do cérebro

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.

A tela de elevada capacidade para Biomining Atividade da celulase de Bibliotecas metagenomic

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.

Transcrição in vitro e nivelamento de Gaussia mRNA luciferase Seguido por HeLa transfecção celular

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.

Cromatina Análise de Interação com Emparelhados Fim-Tag Sequencing (Chia-PET) para mapeamento Interações cromatina e Compreensão regulação da transcrição

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: Epigenoma célula tipo-específicas de criação de perfil

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.

Isolamento de Núcleos do Rim de Rato Adulto para Sequenciamento de RNA de Núcleo Único

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

Isolamento e Perfil de Núcleos Únicos de Tecidos de Mamíferos Congelados

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.

Autor em destaque: Aprimorando a descoberta de medicamentos - Desenvolvimento de protocolos automatizados e padronizados para extração de núcleos de tecidos congelados 

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.

Um Protocolo Simples, Rápido e Parcialmente Automatizado para o Isolamento de Núcleos Únicos de Tecidos Congelados de Mamíferos para Sequenciamento de Núcleo Único