Diese Arbeit wurde durch Mittel aus dem Morgridge Institut für Forschung und der University of Wisconsin-Stiftung unterstützt. Wir danken Krista Eastman für redaktionelle Unterstützung.

<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>

Die Autoren offenbaren, dass JAT einer der Gründer, Aktionärskapitalismus abgelöst, Berater und Vorstandsmitglied von Cellular Dynamics International (CDI) ist. Er dient auch als wissenschaftlicher Berater und finanziellen Interessen in Tactics II Stem Cell Ventures.

Aktuelle Protokolle für die Herstellung gepaart Ende Bibliotheken benötigen zwischen 1 pg 9 bis 2,5 pg 10 ab Höhe der Gesamt-RNA. Hier präsentieren wir unsere lineare T7 Amplifikation (T7LA)-Methode, um sowohl ein Lese-und gepaart Ende Illumina Sequenzierung Bibliotheken und Show vorzubereiten, dass diese Methode ermöglicht die Erstellung von Bibliotheken aus so niedrig wie 10 ng Gesamt-RNA, Herstellung Daten, die vergleichbar mit der ist minimal verstärkt (MinAmp) Bibliotheken von 1000 fold Ausgangsmaterial mehr (10 ug Gesamt-RNA) hergestellt. Die 10 ng Bibliotheken nicht nur identifizieren, ähnlich Gesamtzahl der Gene, sondern auch zu produzieren Genexpressionssignaturen, die ähnlich sind (Abb. 3a und b). Darüber hinaus sind sowohl die einzelnen Lese-und gepaart Ende Bibliotheken durch die T7LA Verfahren hergestellte sehr ähnlich zueinander (Abbildung 3c), denen die Forscher die Daten von Bibliotheken aus beiden Protokolle erwirtschaftet vergleichen können. Da diese Bibliotheken aus humanen embryonalen Stammzellen RNA bereit waren, suchten wirfür 30 Stammzell-spezifische Gene unter den Bibliotheken und feststellen, dass fast alle diese Gene (93-100%) durch die Bibliotheken von mindestens 10 ng Gesamt-RNA ab (Abbildung 4) gemacht werden identifiziert, damit die Validierung unseres Protokolls. Wir glauben, dass unser Protokoll wäre sehr nützlich für die Forscher, besonders in Fällen wie flow sortierten Zellen oder Laser-Mikro-seziert Gewebe, in dem Ausgangsmaterial ist begrenzt. In einem solchen Fall würde unser Protokoll erlauben Generation von Genexpressionsdaten vergleichbar mit Bibliotheken aus viel größeren Mengen ab gemacht, da unser Protokoll produziert Expressionsprofile vergleichbar mindestens 3 Größenordnungen der Ausgangs-RNA.

<table border="1"><tbody><tr><td> Firma </td><td> Kit / Reagent </td><td> Catalog # </td><td> Besondere </td></tr><tr><td> Ambion </td><td> Fragmentation Reagenz </td><td> AM8740 </td><td></td></tr><tr><td> Ambion </td><td> Liner Acrylamid </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> Nicht DEPC behandelt Nuclease freies Wasser </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> methylierte dNTPs </td><td> R0431 </td><td> Für Single Read Probenvorbereitung nur </td></tr><tr><td> Illumina </td><td> TruSeq SR Cluster-Generation kit v5 </td><td> GD-203-5001 </td><td> Für Single Read Probenvorbereitung nur </td></tr><tr><td> Illumina </td><td> TruSeq Seq Kit v5 36 Zyklen </td><td> FC-104-5001 </td><td></td></tr><tr><td> Illumina </td><td> Chip Seq Sample Prep Kit </td><td> IP-102-1001 </td&gt; <td> Für Single Read Probenvorbereitung nur. Kann mit NEB DNA Probenvorbereitung Master Mix ersetzt Set 1 Cat # E6040S </td></tr><tr><td> Illumina </td><td> TruSeq PE Cluster-Generation kit v5 </td><td> PE-203-5001 </td><td> Für gepaart Ende Probenvorbereitung nur </td></tr><tr><td> Illumina </td><td> Gepaart End Sample Prep Kit </td><td> PE-102-1001 </td><td> Für gepaart Ende Probenvorbereitung nur </td></tr><tr><td> Invitrogen </td><td> 1kb Plus Leiter </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> 2. Strang-Puffer 5x </td><td> 10812-014 </td><td></td></tr><tr><td> Invitrogen </td><td> E. coli-DNA-Polymerase </td><td> 18010-017 </td><td></td></tr><tr><td> Invitrogen </td><td> E-Gel SYBR Safe 2% </td><td> G521802 </td><td></td></tr><tr><td> Invitrogen </td><td> Superscript III (mit 5-fach FS-Puffer und 0,1 M DTT) </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 quibit Assay-Kit </td><td> Q32852 </td><td></td></tr><tr><td> Invitrogen </td><td> dsDNA HS Qubit-Assay-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> T4 DNA Polymerase </td><td> 18005-025 </td><td></td></tr><tr><td> Invitrogen </td><td> Ultra Pure Dnase Rnase Wasser </td><td> 10977-015 </td><td></td></tr><tr><td> Invitrogen </td><td> Superscript II doppelsträngigen cDNA-Synthese-Kit </td><td> 11917-020 </td><td></td></tr><tr><td> Invitrogen </td><td> Random Primer (Invitrogen) </td><td> 48190-011 </td><td> Für gepaart Ende Probenvorbereitung nur </td></tr><tr><td> IDT </td><td> Not1Nonamer B Primer </td><td> N / A </td><td> Für Single Read samPLE prep nur </td></tr><tr><td> IDT </td><td> Oligo dT T7 </td><td> N / A </td><td></td></tr><tr><td> NEB </td><td> Not1 Verdauung kit </td><td> R0189S </td><td> Für Single Read Probenvorbereitung nur </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> Gel Purification Kit </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 Forschung </td><td> DNA sauber und Konzentrator (250X) </td><td> D4014 </td><td></td></tr></tbody></table>

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

Whole Transkriptom-Sequenzierung von mRNA-Seq ist nun weitgehend auf globale Genexpression, Mutation, Allel-spezifische Expression und anderen genomweiten Analysen durchzuführen verwendet. mRNA-Seq selbst öffnet das Tor zur Genexpressionsanalyse der nicht-sequenzierten Genomen. mRNA-Seq bietet eine hohe Empfindlichkeit, einen großen Dynamikbereich und erlaubt die Messung der Niederschrift Kopienzahl in einer Probe. Illumina Genome Analyzer führt die Sequenzierung einer großen Anzahl (&gt; 10 7) von relativ kurzer Sequenzabschnitte (&lt;150 bp). Der &quot;paired end&quot;-Ansatz, wobei eine einzelne lange lesen an seinen beiden Enden ist sequenziert, erlaubt für die Verfolgung von alternativen Spleißstellen , Insertionen und Deletionen, und ist für de novo Transkriptom Montage nützlich. Eine der wichtigsten Herausforderungen, denen Forscher gegenüberstehen wird eine begrenzte Menge des Ausgangsmaterials. Zum Beispiel in Experimenten, bei denen Zellen durch Laser-Mikrodissektion, zur Verfügung geerntet werden ab Gesamt-RNA may Maßnahme Nanogramm. Vorbereitung der mRNA-Seq-Bibliotheken aus solchen Proben wurden 1, 2 beschrieben, aber mit erheblichen PCR-Amplifikation, dass Bias einführen kann. Andere RNA-Seq Bibliothek Bauverfahren mit minimalen PCR-Amplifikation wurden 3, 4 veröffentlicht, aber benötigen Mikrogramm-Mengen von Gesamt-RNA ab. Hier beschreiben wir ein Protokoll für den Illumina Genome Analyzer II Plattform für mRNA-Seq-Sequenzierung für die Bibliothek Vorbereitung, die erhebliche PCR-Amplifikation vermeidet und benötigt nur 10 Nanogramm Gesamt-RNA. Während dieses Protokoll wurde zuvor auch für Single-End-Sequenzierung 5, wo sie gezeigt, dass direktionale Bibliotheken, ohne dabei signifikante Verstärkung Bias produzieren beschrieben wurde validiert, hier sind wir zu validieren es weiter für die Verwendung als ein gekoppeltes Ende-Protokoll. Wir selektiv zu verstärken polyadenyliert Boten-RNAs aus beginnend Gesamt-RNA mit der T7 basiert Eberwine lineare Verstärkung Methode geprägt &quot;T7LA &quot;(T7 lineare Verstärkung). Die verstärkten Poly-A-mRNAs fragmentiert sind, revers transkribiert und Adapter ligiert, um die endgültige Sequenzierung Bibliothek zu erzeugen. Für beide gelesen und gepaart Ende läuft, Sequenzen des menschlichen Transkriptom 6 sind abgebildet und so normiert, dass Daten aus mehreren Läufen kann verglichen werden. Wir berichten über die Messung der Genexpression in Einheiten von Transkripten pro Million (TPM), die eine überlegene Maßnahme RPKM beim Vergleich Proben 7 ist.

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

Hier beschreiben wir ein Verfahren zur Herstellung der beiden einzigen gelesen und gepaart Ende Illumina mRNA-Seq-Sequenzierung Bibliotheken zur Genexpressionsanalyse auf T7 lineare RNA-Amplifikation basieren. Dieses Protokoll erfordert nur 10 Nanogramm ab Gesamt-RNA und erzeugt sehr konsistent Bibliotheken für ganze Transkripte.

Single Read und Paired End mRNA-Seq Illumina Bibliotheken von 10 Nanogramm Gesamt-RNA

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 Aufrufe.  Morgridge Institute for Research. Hier beschreiben wir ein Verfahren zur Herstellung der beiden einzigen gelesen und gepaart Ende Illumina mRNA-Seq-Sequenzierung Bibliotheken zur Genexpressionsanalyse auf T7 lineare RNA-Amplifikation basieren. Dieses Protokoll erfordert nur 10 Nanogramm ab Gesamt-RNA und erzeugt sehr konsistent Bibliotheken für ganze Transkripte.

Serie: 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.

Large-Scale-Bildschirme von Metagenombanken

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

Forschung

Biologie

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

RNA Extraktion aus Neuroprecursor Zellen unter Verwendung des Bio-Rad Total 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.

Beziehen High Quality-RNA aus Einzel-Zellpopulationen in menschlichen Postmortem Gehirngewebe

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.

Ein High-Throughput-Screen für Biomining Cellulase Aktivität von Metagenombanken

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.

In vitro Transkription und Capping von Gaussia-Luciferase mRNA von HeLa-Zelle Transfektion Gefolgt

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.

Chromatin Interaction Analysis gepaart mit End-Tag-Sequenzierung (Chia-PET) zur Abbildung von Chromatin-Wechselwirkungen zu verstehen und Transkriptionsregulation

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: Cell-Type-Specific Epigenom Profilierung

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.

Zellkernisolierung aus der Niere adulter Mäuse für die Einzelkern-RNA-Sequenzierung

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

Isolierung und Profilierung einzelner Zellkerne aus gefrorenem Säugetiergewebe

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.

Author Spotlight: Enhancing Drug Discovery - Entwicklung automatisierter, standardisierter Protokolle für die Zellkernextraktion aus gefrorenem Gewebe 

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.

Ein einfaches, schnelles und teilautomatisiertes Protokoll für die Isolierung von Einzelkernen aus gefrorenem Säugetiergewebe für die Einzelkernsequenzierung