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	<li>Addepalli, B., Venus, S., Thakur, P., Limbach, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+ribonuclease+activity+of+cusativin+from+Cucumis+sativus+for+mapping+nucleoside+modifications+in+RNA.">Novel ribonuclease activity of cusativin from Cucumis sativus for mapping nucleoside modifications in RNA.</a> Analytical and Bioanalytical Chemistry. 409 (24), 5645-5654 (2017).</li><li>Gao, H., Liu, Y., Rumley, M., Yuan, H., Mao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+confirmation+of+chemically+modified+RNAs+using+exonuclease+digestion+and+matrix-assisted+laser+desorption/ionization+time-of-flight+mass+spectrometry.">Sequence confirmation of chemically modified RNAs using exonuclease digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 23 (21), 3423-3430 (2009).</li><li>McLuckey, S. A., Van Berkel, G. J., Glish, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+mass+spectrometry+of+small,+multiply+charged+oligonucleotides.">Tandem mass spectrometry of small, multiply charged oligonucleotides.</a> Journal of The American Society for Mass Spectrometry. 3 (1), 60-70 (1992).</li><li>Fountain, K. J., Gilar, M., Gebler, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+native+and+chemically+modified+oligonucleotides+by+tandem+ion-pair+reversed-phase+high-performance+liquid+chromatography/electrospray+ionization+mass+spectrometry.">Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 17 (7), 646-653 (2003).</li><li>Taucher, M., Breuker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+modified+RNA+by+top-down+mass+spectrometry.">Characterization of modified RNA by top-down mass spectrometry.</a> Angewandte Chemie International Edition in English. 51 (45), 11289-11292 (2012).</li><li>Kellner, S., Burhenne, J., Helm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+RNA+modifications.">Detection of RNA modifications.</a> RNA Biology. 7 (2), 237-247 (2010).</li><li>Thomas, B., Akoulitchev, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+of+RNA.">Mass spectrometry of RNA.</a> Trends in Biochemical Sciences. 31 (3), 173-181 (2006).</li><li>Bjorkbom, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+direct+sequencing+of+noncanonical+RNA+by+two-dimensional+analysis+of+mass+chromatograms.">Bidirectional direct sequencing of noncanonical RNA by two-dimensional analysis of mass chromatograms.</a> Journal of the American Chemical Society. 137 (45), 14430-14438 (2015).</li><li>Zhang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+LC-MS-based+RNA+sequencing+method+for+direct+analysis+of+multiple-base+modifications+in+RNA+mixtures.">A general LC-MS-based RNA sequencing method for direct analysis of multiple-base modifications in RNA mixtures.</a> Nucleic Acids Research. 47 (20), 125 (2019).</li><li>Zhang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+sequencing+of+tRNA+by+2D-HELS-AA+MS+Seq+reveals+its+different+isoforms+and+dynamic+base+modifications.">Direct sequencing of tRNA by 2D-HELS-AA MS Seq reveals its different isoforms and dynamic base modifications.</a> ACS Chemical Biology. 15 (6), 1464-1472 (2020).</li><li>Bakin, A., Ofengand, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+newly+located+pseudouridylate+residues+in+Escherichia+coli+23S+ribosomal+RNA+are+all+at+the+peptidyltransferase+center:+analysis+by+the+application+of+a+new+sequencing+technique.">Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique.</a> Biochemistry. 32 (37), 9754-9762 (1993).</li><li>Cantara, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+Modification+Database,+RNAMDB:+2011+update.">The RNA Modification Database, RNAMDB: 2011 update.</a> Nucleic Acids Research. 39 (Database issue), D195-D201 (2011).</li></ol>

<table border="0" cellpadding="0" cellspacing="0" style="width:715px;" width="714">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:227px;">5&#39; DNA Adenylation kit</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:156px;">E2610S</td>
			<td style="width:167px;">50uM concentration</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">6550 Q-TOF mass spectrometer</td>
			<td>Agilent Technologies</td>
			<td style="width:156px;">5991-2116EN</td>
			<td>Coupled to a 1290 Infinity LC system</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">A(5&#180;)pp(5&#180;)Cp-TEG-biotin-3&#180;</td>
			<td>ChemGenes</td>
			<td>91718</td>
			<td>HPLC purified</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ATP&#947;S</td>
			<td>Sigma-Aldrich</td>
			<td>11162306001</td>
			<td>Lithium salt</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bicine</td>
			<td>Sigma-Aldrich</td>
			<td>B8660</td>
			<td>BioXtra, &#8805;99% (titration)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Biotin maleimide</td>
			<td>Vector Laboratories</td>
			<td>SP-1501</td>
			<td>Long arm</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">C18 column</td>
			<td>Waters</td>
			<td style="width:156px;">186003532</td>
			<td>50 mm &#215; 2.1 mm Xbridge C18 column with a particle size of 1.7 &#956;m</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Centrifugal Vacuum Concentrator</td>
			<td>Labconco</td>
			<td>Refrig 115v/60hz 7310022</td>
			<td>Labconco CentriVap</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ChemBioDraw</td>
			<td>PerkinElmer</td>
			<td>ChemDraw Prime</td>
			<td>Generate a chemical structure and property data of structures &#38; fragments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">CMC (N-cyclohexyl-N&#697;-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate)</td>
			<td>Sigma-Aldrich</td>
			<td>2491-17-0</td>
			<td>95% Purifiy</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cyanine3 maleimide (Cy3)</td>
			<td>Lumiprobe</td>
			<td>11080</td>
			<td>Water insoluble</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DEPC-treated water</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9906</td>
			<td>Autoclaved, certified nuclease-free</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Diisopropylamine (DIPA)</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:156px;">108-18-9</td>
			<td>99% Alfa Aesar</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td>Anhydrous dimethyl sulfoxide, 99.9%</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td>Anhydrous, crystalline, BioReagent, suitable for cell culture</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Formic acid</td>
			<td>Merck</td>
			<td>64-18-6</td>
			<td>98-100%, ACS reag, Ph Eur</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hexafluoro-2-propanol (HFIP)</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:156px;">920-66-1</td>
			<td>99% Acros Organics</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LC-MS sample vials</td>
			<td>Thermo Fisher Scientific</td>
			<td>C4000-11</td>
			<td>Plastic screw thread vials</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LC-MS vial caps</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:156px;">C5000-54A</td>
			<td>Autosampler vial screw thread caps</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Na2CO3 buffer</td>
			<td>Sigma-Aldrich</td>
			<td>88975</td>
			<td>BioUltra, &#62;0.1 M Na2CO3, &#62;0.2 M NaHCO3</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Oligo Clean &#38; Concentrator</td>
			<td>Zymo Research</td>
			<td style="width:156px;">D4060</td>
			<td>Spin column</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">OriginLab</td>
			<td>OriginLab</td>
			<td>OriginPro</td>
			<td>Data analysis and graphing software</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">pCp-biotin</td>
			<td>TriLink BioTechnologies</td>
			<td>NU-1706-BIO</td>
			<td>20 ul (1 mM)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RNA #1--#6</td>
			<td>Integrated DNA Technologies</td>
			<td>Custom RNA oligos</td>
			<td>19nt-21nt single-stranded RNAs, used without further purification</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rocking platform shaker</td>
			<td>VWR</td>
			<td>Orbital Shaker Standard 1000</td>
			<td>Speed Range 40 to 300 rpm</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Streptavidin magnetic beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>88816</td>
			<td>Binding approx. 55ug biotinylated rabbit lgG per mg of beads</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sulfonated Cyanine3 maleimide</td>
			<td>Lumiprobe</td>
			<td>11380</td>
			<td>Water soluble</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">T4 DNA ligase 1</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td>400 units/uL</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">T4 polynucleotide kinase</td>
			<td>Sigma-Aldrich</td>
			<td>T4PNK-RO</td>
			<td>From phage T4 am N81 pse T1 infected Escherichia coli BB</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl buffer</td>
			<td>Sigma-Aldrich</td>
			<td>T6455</td>
			<td>Tris-HCl Buffer, pH 10, 10&#215;, Antigen Retriever</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Urea</td>
			<td>Sigma-Aldrich</td>
			<td>81871</td>
			<td>Urea for synthesis. CAS No. 57-13-6, EC Number 200-315-5.</td>
		</tr>
	</tbody>
</table>

2d-hels ms seq: a general lc-ms-based method for direct and de novo sequencing of rna mixtures with different nucleotide modifications

Watch this Scientific Journal Video about 2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications at JoVE.com

<meta charset="utf8"></head><body>2D-HELS MS Seq: Un metodo generale basato su LC-MS per il sequenziamento diretto e de novo di miscele di RNA con diverse modifiche nucleotidiche

2D-HELS MS Seq: Un metodo generale basato su LC-MS per il sequenziamento diretto e de novo di miscele di RNA con diverse modifiche nucleotidiche

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

2d-hels-ms-seq-general-lc-ms-based-method-for-direct-de-novo

Research

JoVE Journal

Genetics

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

1.8K Views.  New York Institute of Technology. Qui, descriviamo un protocollo dettagliato per un metodo di sequenziamento basato su LC-MS che può essere utilizzato come metodo diretto per sequenziare RNA corto (

Video: 2D-HELS MS Seq: Un metodo generale basato su LC-MS per il sequenziamento diretto e de novo di miscele di RNA con diverse modifiche nucleotidiche

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved (Non-model) Organisms

This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative transcriptomics. It also serves as a primer for biologists to develop data scientist computational skills, e.g. executing bash commands, visualization and management of large data sets. All command line code and further explanations of each command or step can be found on the wiki (<a href="https://wiki.cyverse.org/wiki/x/dgGtAQ">https://wiki.cyverse.org/wiki/x/dgGtAQ</a>). The Discovery Environment and Atmosphere platforms are connected together through the CyVerse Data Store. As such, once the initial raw sequencing data has been uploaded there is no more need to transfer large data files over an Internet connection, minimizing the amount of time needed to conduct analyses. This protocol is designed to analyze only two experimental treatments or conditions. Differential gene expression analysis is conducted through pairwise comparisons, and will not be suitable to test multiple factors. This workflow is also designed to be manual rather than automated. Each step must be executed and investigated by the user, yielding a better understanding of data and analytical outputs, and therefore better results for the user. Once complete, this protocol will yield de novo assembled transcriptome(s) for underserved (non-model) organisms without the need to map to previously assembled reference genomes (which are usually not available in underserved organism). These de novo transcriptomes are further used in pairwise differential gene expression analysis to investigate genes differing between two experimental conditions. Differentially expressed genes are then functionally annotated to understand the genetic response organisms have to experimental conditions. In total, the data derived from this protocol is used to test hypotheses about biological responses of underserved organisms.

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved Non-model Organisms

This protocol outlines a comparative de novo transcriptome assembly and annotation workflow for novice bioinformaticians. The workflow is available for free entirely through CyVerse and connected by the Data Store. Command line and graphical user interfaces are used, but all code needed is available to copy and paste.

Utilizzare risorse CyVerse per<em&gt; De Novo</em&gt; Transcriptomics comparativi degli organismi non sottoposti a modellazione (non modello)

This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative ...

Ricerca

Genetica

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging resistance mutations during therapy, or when patients have few circulating tumor cells. Wild-type blocking PCR followed by sequencing analysis offers high sensitivity, flexibility, and simplicity as a methodology for detecting these low frequency mutations. By adding a custom designed locked nucleic acid oligonucleotide to a new or previously established conventional PCR based sequencing assay, sensitivities of approximately 1 mutant allele in a background of 1,000 WT alleles can be achieved (1:1,000). Sequencing artifacts associated with deamination events commonly found in formalin fixed paraffin embedded tissues can be partially remedied by the use of uracil DNA glycosylase during extraction steps. The optimized protocol here is specific for detecting MYD88 mutation, but can serve as a template to design any WTB-PCR assay. Advantages of the WTB-PCR assay over other commonly utilized assays for the detection of low frequency mutations including allele specific PCR and real-time quantitative PCR include fewer occurrences of false positives, greater flexibility and ease of implementation, and the ability to detect both known and unknown mutations.

Wild-type blocking PCR followed by direct sequencing offers a highly sensitive method of detection for low frequency somatic mutations in a variety of sample types.

Wild-type Blocco PCR Combinato con sequenziamento diretto come un metodo altamente sensibile per il rilevamento di bassa frequenza di mutazioni somatiche

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

G2-seq: Un'elevato Throughput sequenziamento-base tecnica per identificare replica ritardata regioni del genoma

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Rilevamento di eventi rari utilizzando la sequenza di RNA e DNA di correzione d'errore

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

Produzione di topi transgenici mediata lentivirali: un metodo semplice ed altamente efficiente per uno studio diretto dei fondatori

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Saccharomyces cerevisiae Metabolica etichettatura con 4-tiouracile e la quantificazione di nuova sintesi mRNA come Proxy per attività di RNA polimerasi II

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

Identificazione di High-throughput di sequenze regolatorie del Gene mediante sequenziamento di nuova generazione di cromosoma circolare conformazione catturare (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

Identificazione delle impronte di RNA:Complessi proteici tramite l'immunoprecipitazioni dell'RNA in Tandem seguita dal sequenziamento (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

A Method to Study de novo Formation of Chromatin Domains

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity and/or disease. Despite tremendous efforts, our understanding of the formation and propagation of chromatin domains is still limited. Chromatin domains have been studied under steady-state conditions, which are not conducive to following the initial events during their establishment. Here, we present a method to inducibly reconstruct chromatin domains and follow their re-formation as a function of time. Although, first applied to the case of PRC2-mediated repressive chromatin domain formation, it could be easily adapted to other chromatin domains. The modification of and/or the combination of this method with genomics and imaging technologies will provide invaluable tools to study the establishment of chromatin domains in great detail. We believe that this method will revolutionize our understanding of how chromatin domains form and interact with each other.

A Method to Study de novo Formation of Chromatin Domains

This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems.

Un metodo per studiare la formazione de novo dei domini della cromatina

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity ...

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

Migliorare il piccolo RNA-seq: meno Bias e migliore rilevamento di 2'-O-Methyl RNA

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...