עבודה זו נתמכה על ידי מימון ממכון Morgridge למחקר באוניברסיטת ויסקונסין קרן. אנו מודים קריסטה איסטמן לסיוע העריכה.

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	<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>

החוקרים לגלות כי ג&#39;ת הוא מייסד, stockowner, יועץ וחבר מועצת המנהלים של נייד Dynamics International (CDI). הוא גם משמש כיועץ מדעי ויש לו אינטרסים פיננסיים טקטיקות תא גזע ונצ&#39;רס II.

פרוטוקולים נוכחי להכנת ספריות סוף לזווג דורשים בין 1 מיקרוגרם 9-2.5 10 מיקרוגרם סכום התחלתי של RNA מוחלט. כאן אנו מציגים T7 מבוסס ליניארי שלנו הגברה (T7LA) שיטה להכין גם אחד לקרוא לזווג Illumina סוף ספריות רצף ולהראות כי שיטה זו מאפשרת לדור של ספריות מ ng נמוך כמו 10 של רנ&quot;א הכל, הפקת נתונים דומה לזה של הגברה מינימלית (MinAmp) ספריות עשוי מחומר 1000 החל לקפל יותר (10 מיקרוגרם RNA סה&quot;כ). 10 ספריות ng לא רק לזהות את המספרים הכוללים דומה של גנים, אלא גם לייצר חתימות התבטאות גנים דומים (איורים 3 א ו - ב). יתר על כן, גם ספריות יחיד סוף לקרוא לזווג המיוצר על ידי השיטה T7LA דומים מאוד זה לזה (איור 3c), אשר מאפשרת לחוקרים להשוות נתונים שנוצרו על ידי ספריות עשויות גם פרוטוקול. מכיוון שמדובר בספריות היו מוכנים מן האדם RNA לתא גזע עובריים, חיפשנועבור 30 גנים תא גזע מסוים בין הספריות למצוא שכמעט כל הגנים האלה (93-100%) מזוהים הספריות עשוי לפחות 10 ng להתחיל RNA הכולל (איור 4), ובכך אימות פרוטוקול שלנו. אנו מאמינים פרוטוקול שלנו יהיה מאוד שימושי עבור חוקרים, במיוחד בנסיבות כגון תאים זרימת מיון או לייזר מיקרו גזור רקמת שם חומר המוצא היא מגבילה. בנסיבות כאלה, פרוטוקול שלנו תאפשר הדור של נתונים ביטוי גנים דומים ספריות עשוי כמויות הרבה יותר גדול מאז החל פרוטוקול שלנו מייצרת פרופילי ביטוי דומה על פני לפחות 3 סדרי גודל של רנ&quot;א ההתחלה.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> חברה </td><td> קיט / מגיב </td><td> קטלוג # </td><td> מיוחד תגובות </td></tr><tr><td> Ambion </td><td> פיצול מגיב </td><td> AM8740 </td><td></td></tr><tr><td> Ambion </td><td> Liner Acrylamide </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 ללא טיפול במים nuclease חינם </td><td> AM9932 </td><td></td></tr><tr><td> Fermentas </td><td> dNTP להגדיר </td><td> R0181 </td><td></td></tr><tr><td> Fermentas </td><td> מפוגל dNTPs </td><td> R0431 </td><td> עבור הכנה יחיד מדגם לקריאה בלבד </td></tr><tr><td> Illumina </td><td> TruSeq SR אשכול הדור ערכת v5 </td><td> GD-203-5001 </td><td> עבור הכנה יחיד מדגם לקריאה בלבד </td></tr><tr><td> Illumina </td><td> TruSeq Seq ערכת v5 36 מחזורים </td><td> FC-104-5001 </td><td></td></tr><tr><td> Illumina </td><td> שבב Seq מדגם Prep Kit </td><td> IP-102-1001 </td&gt; <td> עבור הכנה יחיד מדגם קריאה בלבד. יכול להיות מוחלף עם חוד מערבבים DNA הכנה המדגם מאסטר סט 1 # חתול E6040S </td></tr><tr><td> Illumina </td><td> TruSeq PE אשכול הדור ערכת v5 </td><td> PE-203-5001 </td><td> עבור הכנה לזווג מדגם רק בסוף </td></tr><tr><td> Illumina </td><td> מותאמים לדוגמא סוף Prep Kit </td><td> PE-102-1001 </td><td> עבור הכנה לזווג מדגם רק בסוף </td></tr><tr><td> Invitrogen </td><td> 1kb סולם בתוספת </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> גדיל חוצץ 2 5x </td><td> 10812-014 </td><td></td></tr><tr><td> Invitrogen </td><td> E. coli DNA פולימראז </td><td> 18010-017 </td><td></td></tr><tr><td> Invitrogen </td><td> E-ג&#39;ל SYBR% בטוח 2 </td><td> G521802 </td><td></td></tr><tr><td> Invitrogen </td><td> כתב עילי אניהשני (עם 5X FS חיץ 0.1M 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 ערכת </td><td> Q32852 </td><td></td></tr><tr><td> Invitrogen </td><td> dsDNA HS קיוביט assay ערכת </td><td> Q32851 </td><td></td></tr><tr><td> Invitrogen </td><td> E. coli האנזים DNA </td><td> 18052-019 </td><td></td></tr><tr><td> Invitrogen </td><td> T4 DNA פולימראז </td><td> 18005-025 </td><td></td></tr><tr><td> Invitrogen </td><td> אולטרה טהורים מים Dnase Rnase </td><td> 10977-015 </td><td></td></tr><tr><td> Invitrogen </td><td> כתב עילי כפול II תקועים ערכת סינתזה cDNA </td><td> 11917-020 </td><td></td></tr><tr><td> Invitrogen </td><td> פריימר אקראית (Invitrogen) </td><td> 48190-011 </td><td> עבור הכנה לזווג מדגם רק בסוף </td></tr><tr><td> IDT </td><td> Not1Nonamer תחל ב </td><td> N / A </td><td> עבור יחיד קראו סםple הכנה בלבד </td></tr><tr><td> IDT </td><td> אוליגו DT T7 </td><td> N / A </td><td></td></tr><tr><td> חוד </td><td> Not1 העיכול ערכת </td><td> R0189S </td><td> עבור הכנה יחיד מדגם לקריאה בלבד </td></tr><tr><td> Qiagen </td><td> Rneasy Minelute ערכת </td><td> 74204 </td><td></td></tr><tr><td> Qiagen </td><td> RNEasy מיני קיט (50) </td><td> 74104 </td><td></td></tr><tr><td> Qiagen </td><td> ג&#39;ל לטיהור ערכת </td><td> 28604 </td><td></td></tr><tr><td> Qiagen </td><td> Dnase להגדיר </td><td> 79254 </td><td></td></tr><tr><td> Qiagen </td><td> Rneasy MinElute ערכת </td><td> 74204 </td><td></td></tr><tr><td> Zymo מחקר </td><td> ה-DNA נקי concentrator (250X) </td><td> D4014 </td><td></td></tr></tbody></table>

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

רצף transcriptome שלמות על ידי Seq-mRNA משמש כיום באופן נרחב לבצע ביטוי גנים מוטציה העולמי, ספציפית אלל הביטוי ושאר הגנום כולו מנתח. mRNA-Seq אפילו פותח את השער לצורך ניתוח גנטי הביטוי של אי - רצף הגנום. mRNA-Seq מציע רגישות גבוהה, טווח דינמי גדול המאפשר מדידה של מספרים עותק תמליל במדגם. הגנום של Illumina מנתח מבצע רצף של מספר גדול (&gt; 10 7) של רצף קצר יחסית קורא (&lt;150 נ&quot;ב). &quot;לזווג סוף&quot; הגישה, לפיה לקרוא ארוך אחד הוא רצף בשני הקצוות שלה, מאפשרת מעקב בצמתים אחוי חלופי , הוספות ומחיקות, והוא שימושי להרכבה transcriptome דה נובו. אחד האתגרים העיקריים העומדים בפני החוקרים היא כמות מוגבלת של חומר המוצא. לדוגמה, בניסויים שבהם התאים נקצרים על ידי לייזר מיקרו לנתיחה, הכל זמין החל מ-RNAאיי למדוד ננוגרם. הכנת ה-mRNA-Seq ספריות דגימות כאלה תוארו 1, 2, אך כרוך הגברה משמעותית PCR שעשויים להציג את ההטיה. אחרים RNA-Seq הליכי הבנייה ספריה עם הגברה PCR מינימלי פורסמו 3, 4, אבל דורשים כמויות מיקרוגרם של החל RNA מוחלט. כאן אנו מתארים פרוטוקול עבור פלטפורמת הגנום Illumina השנייה Analyzer עבור סידור-mRNA Seq להכנת ספריה נמנע משמעותי הגברה PCR דורש רק 10 ננוגרם של רנ&quot;א הכל. אמנם זה פרוטוקול תוארה בעבר תוקף עבור יחיד סוף רצף 5, שם הוצגה לייצר ספריות כיוונית ללא החדרת הטיה הגברה משמעותית, הנה אנחנו לאמת אותו עוד יותר לשימוש בפרוטוקול סוף לזווג. אנחנו סלקטיבי להגביר RNAs שליח polyadenylated החל מ RNA סך באמצעות שיטה המבוססת T7 Eberwine הגברה ליניארית, שטבע &quot;T7LA &quot;(הגברה T7 ליניארי). מוגבר מרובה mRNAs הם מפוצלים, הפוכה יתוכתבו מתאם ligated לייצר ספריית רצף הסופי. עבור שתי ריצות יחיד סוף לקרוא יחד, רצפים ממופים transcriptome האנושי 6 ו מנורמל כך נתוני פועל מרובים ניתן להשוות. אנחנו הדו&quot;ח ביטוי גנים המדידה ביחידות של תמלילי למיליון (TPM), המהווה מדד מעולה RPKM כאשר משווים דגימות 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

כאן אנו מתארים שיטה להכנת שני יחיד לקרוא לזווג Illumina סוף mRNA-Seq ספריות לניתוח רצפי גנים הביטוי מבוסס על הגברה T7-RNA ליניארי. פרוטוקול זה דורש רק 10 ננוגרם של רנ&quot;א הכל החל ומייצר ספריות עקבי מאוד המייצגים תמלילי כולו.

יחיד קרא מותאמים mRNA-Seq סוף Illumina ספריות מ -10 ננוגרם RNA סך הכל

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

Nanyang Technological University

Research

JoVE Journal

Biology

39.1K Views.  Morgridge Institute for Research. כאן אנו מתארים שיטה להכנת שני יחיד לקרוא לזווג Illumina סוף mRNA-Seq ספריות לניתוח רצפי גנים הביטוי מבוסס על הגברה T7-RNA ליניארי. פרוטוקול זה דורש רק 10 ננוגרם של רנ"א הכל החל ומייצר ספריות עקבי מאוד המייצגים תמלילי כולו.

Video: 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.

בקנה מידה גדול של מסכי ספריות Metagenomic

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

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

הפקת RNA מן התאים Neuroprecursor שימוש Bio-Rad סך 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.

קבלת RNA איכות גבוהה מאוכלוסיות תא בודד בתוך רקמות המוח האנושי שלאחר המוות

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.

מסך התפוקה גבוהה עבור Biomining פעילות cellulase מספריות Metagenomic

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

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

מסכי הגנום תחרותי של ספריות שמרים המבורקדים

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

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.

בתעתיק במבחנה תקרת של mRNA בלוציפראז Gaussia ואחריו transfection Cell הלה

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.

הכרומטין ניתוח אינטראקציה עם מנבא סוף תג הרצף (Chia-PET) עבור מיפוי אינטראקציות הכרומטין הבנת תקנה תמלול

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

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

ניתוח של תגובות RNA עיבוד באמצעות מערכות חינם ניידים: 3 &#39;סוף המחשוף של Pre-mRNA מצעים<em&gt; במבחנה</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

ניתוח של גלובל RNA סינתזה ברמת התא בודד הבאות היפוקסיה

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

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: יצירת פרופילים ייחודיים לסוג התא Epigenome

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