이 작품은 연구와 위스콘신 재단의 대학 Morgridge 연구소의 자금 지원에 의해 지원되었다. 우리는 편집 지원 크리스타 이스트만 주셔서 감사합니다.

<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>

저자는 잣은 세포 역학 국제 (CDI)의 설립자, stockowner, 컨설턴트 및 이사회의 구성원임을 공개. 그는 또한 과학 고문 역할과 전술 II의 줄기 세포 벤처 금융 이익 있습니다.

Current protocols for making paired end libraries require between 1 &mu;g9 to 2.5 &mu;g10 starting amount of total RNA. Here we present our linear T7 amplification based (T7LA) method to prepare both single read and paired end Illumina sequencing libraries and show that this method allows generation of libraries from as low as 10 ng of total RNA, producing data that is comparable to that of minimally amplified (MinAmp) libraries made from 1000 fold more starting material (10 &mu;g total RNA). The 10 ng libraries not only identify similar total numbers of genes, but also produce gene expression signatures that are similar (Figures 3a and b). Moreover, both the single read and paired end libraries produced by the T7LA method are very similar to each other (Figure 3c), which allows researchers to compare data generated by libraries made from either protocol. Since these libraries were prepared from human embryonic stem cell RNA, we searched for 30 stem cell specific genes among the libraries and find that almost all of these genes (93-100%) are identified by the libraries made from at least 10 ng of starting total RNA (Figure 4), thus validating our protocol. We believe our protocol would be very useful for researchers, especially in circumstances such as flow sorted cells or laser-micro-dissected tissue where starting material is limiting. In such circumstances, our protocol would allow generation of gene expression data comparable to libraries made from much larger starting quantities since our protocol produces expression profiles comparable across at least 3 orders of magnitude of starting RNA.

<table border="1"><tbody><tr><td> 회사 </td><td> 키트 / 시약 </td><td> 카탈로그 # </td><td> 특별 코멘트 </td></tr><tr><td> Ambion </td><td> 파쇄 시약 </td><td> AM8740 </td><td></td></tr><tr><td> Ambion </td><td> 라이너 아크릴 아미드 </td><td> AM9520 </td><td></td></tr><tr><td> Ambion </td><td> MEGAscript T7 키트 </td><td> AM1334 </td><td></td></tr><tr><td> Ambion </td><td> 비 DEPC는 nuclease 무료로 물을 처리 </td><td> AM9932 </td><td></td></tr><tr><td> Fermentas </td><td> dNTP 세트 </td><td> R0181 </td><td></td></tr><tr><td> Fermentas </td><td> methylated dNTPs </td><td> R0431 </td><td> 단일 읽기 샘플 준비에 대해서만 </td></tr><tr><td> Illumina </td><td> TruSeq SR 클러스터 생성 키트 V5 </td><td> GD - 203-5001 </td><td> 단일 읽기 샘플 준비에 대해서만 </td></tr><tr><td> Illumina </td><td> TruSeq Seq 키트 V5 36주기 </td><td> FC - 104-5001 </td><td></td></tr><tr><td> Illumina </td><td> 칩 Seq 샘플 준비 키트 </td><td> IP - 102-1001 </td&gt; <td> 단일 읽기 샘플 준비에 대해서만. 코 DNA 샘플 준비 마스터 믹스로 교체할 수 있습니다 한 고양이 # E6040S 설정 </td></tr><tr><td> Illumina </td><td> TruSeq PE 클러스터 생성 키트 V5 </td><td> PE - 203-5001 </td><td> 이점 엔드 샘플 준비에 대해서만 </td></tr><tr><td> Illumina </td><td> 이점 최종 샘플 준비 키트 </td><td> PE - 102-1001 </td><td> 이점 엔드 샘플 준비에 대해서만 </td></tr><tr><td> Invitrogen </td><td> 1킬로바이트 플러스 사다리 </td><td> 10787-018 </td><td></td></tr><tr><td> Invitrogen </td><td> E. 대장균 Rnase H </td><td> 18021-014 </td><td></td></tr><tr><td> Invitrogen </td><td> Rnase 아웃 </td><td> 10777-019 </td><td></td></tr><tr><td> Invitrogen </td><td> 둘째 스트랜드 버퍼 5 배 </td><td> 10812-014 </td><td></td></tr><tr><td> Invitrogen </td><td> E. 대장균의 DNA 효소 </td><td> 18010-017 </td><td></td></tr><tr><td> Invitrogen </td><td> E - 젤 SYBR 안전 2퍼센트 </td><td> G521802 </td><td></td></tr><tr><td> Invitrogen </td><td> 윗첨자 III (5 배 FS 버퍼와 0.1M DTT 포함) </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 분석 키트 </td><td> Q32852 </td><td></td></tr><tr><td> Invitrogen </td><td> dsDNA HS qubit 분석 키트 </td><td> Q32851 </td><td></td></tr><tr><td> Invitrogen </td><td> E. 대장균 DNA ligase </td><td> 18052-019 </td><td></td></tr><tr><td> Invitrogen </td><td> T4의 DNA 중합 효소 </td><td> 18005-025 </td><td></td></tr><tr><td> Invitrogen </td><td> 울트라 퓨어 Dnase Rnase 물 </td><td> 10977-015 </td><td></td></tr><tr><td> Invitrogen </td><td> 윗첨자 II 이중 좌초 cDNA 합성 키트 </td><td> 11917-020 </td><td></td></tr><tr><td> Invitrogen </td><td> 임의 프라이머 (invitrogen) </td><td> 48190-011 </td><td> 이점 엔드 샘플 준비에 대해서만 </td></tr><tr><td> IDT </td><td> Not1Nonamer B의 입문서 </td><td> N / A </td><td> 단일 읽어 샘ple 준비에만 </td></tr><tr><td> IDT </td><td> Oligo DT T7 </td><td> N / A </td><td></td></tr><tr><td> 코 </td><td> Not1 소화 키트 </td><td> R0189S </td><td> 단일 읽기 샘플 준비에 대해서만 </td></tr><tr><td> Qiagen </td><td> Rneasy Minelute 키트 </td><td> 74,204 </td><td></td></tr><tr><td> Qiagen </td><td> RNEasy 미니 키트 (50) </td><td> 74,104 </td><td></td></tr><tr><td> Qiagen </td><td> 겔 정화 키트 </td><td> 28,604 </td><td></td></tr><tr><td> Qiagen </td><td> Dnase 세트 </td><td> 79,254 </td><td></td></tr><tr><td> Qiagen </td><td> Rneasy MinElute 키트 </td><td> 74,204 </td><td></td></tr><tr><td> Zymo 연구 </td><td> DNA 깨끗하고 농축기 (250X) </td><td> D4014 </td><td></td></tr></tbody></table>

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

mRNA - Seq하여 전체 transcriptome 배열은 현재 전세계 유전자 발현, 돌연변이, allele 특정 표현과 다른 게놈 차원의 분석을 수행하기 위해 광범위하게 사용됩니다. mRNA - Seq도 아닌 합성의 genomes의 유전자 발현 분석을위한 게이트가 열립니다. mRNA - Seq는 높은 감도, 넓은 동적 범위를 제공하고 샘플에 성적표 사본 번호를 측정하실 수 있습니다. Illumina의 게놈 분석기는 하나의 긴 읽기 궁극적으로 &quot;이점 엔드&quot;접근 방식이 모두의 끝에 합성 수 있습니다. 대형 상대적으로 짧은 시퀀스 번호 (&gt; 10 7) (&lt;150 BP) 읽기의 순서 수행 대체 스플 라이스 분기점을 추적 가능 , 삽입 및 삭제, 그리고 드 노보 transcriptome 조립하는 데 유용합니다. 연구자들이 직면한 주요 도전 과제 중 하나는 재료를 시작하는 제한된 금액을 말합니다. 예를 들어, 세포가 레이저 마이크로 해부 가능하여 수확 아르 실험 총 RNA m를 시작나노 그램의 대책을 불안. 이러한 샘플에서 mRNA - Seq 라이브러리의 준비는 1, 2를 설명하지만 편견을 소개 중대한 PCR 증폭을 포함하고 있습니다. 최소한의 PCR 증폭 다른 RNA - Seq 라이브러리 구축 절차 3, 4를 발표했지만 전체 RNA를 시작하는 마이크로 그램 금액을 요구했습니다. 여기 우리는 중요한 PCR 증폭을 피할 수 및 총 RNA의 10 나노 그램이 필요 도서관 준비 mRNA - Seq의 시퀀싱을위한 Illumina 게놈 분석기 II 플랫폼을위한 프로토콜을 설명합니다. 이 프로토콜은 그것이 상당한 증폭 바이어스를 도입하지 않고 방향 라이브러리를 제작 게재된 단일 엔드 시퀀싱 5, 이전에 설명한 및 검증되어 있지만, 여기서 우리는 이점 엔드 프로토콜로 사용하기 위해 추가로 그것을 확인합니다. 우리는 선택 &#39;T7 주장, T7 기반 Eberwine 선형 증폭 방법을 사용하여 총 RNA를 시작 polyadenylated 메신저 RNAs를 증폭라 &quot;(T7 선형 증폭). 증폭 폴리 - A mRNAs이 조각되어 최종 시퀀싱 라이브러리를 생성하는 출혈도 잡았 베꼈는데와 어댑터 역방향. 모두 하나의 읽기 및 이점 최종 실행을 위해, 시퀀스는 인간의 transcriptome 6 매핑하고되도록 정규화 여러 실행에서 데이터를 비교할 수 있습니다. 우리는 샘플 7 비교하면 RPKM에 뛰어난 측정합니다 만명 당 성적 단위로 유전자 발현 측정 (TPM)를보고합니다.

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

여기 T7 RNA 선형 증폭을 기반으로 유전자 발현 분석을 위해 모두 하나의 읽기 및 이점 엔드 Illumina mRNA - Seq 시퀀싱 라이브러리의 준비를위한 방법을 설명합니다. 이 프로토콜은 총 RNA를 시작하는 10 나노 그램이 필요하고 전체 성적표를 나타내는 매우 일관된 라이브러리를 생성합니다.

10 나노 그램 총 RNA의 단일 읽기와 결합하여 최종 mRNA - Seq의 Illumina 도서관

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 조회수.  Morgridge Institute for Research. 여기 T7 RNA 선형 증폭을 기반으로 유전자 발현 분석을 위해 모두 하나의 읽기 및 이점 엔드 Illumina mRNA - Seq 시퀀싱 라이브러리의 준비를위한 방법을 설명합니다. 이 프로토콜은 총 RNA를 시작하는 10 나노 그램이 필요하고 전체 성적표를 나타내는 매우 일관된 라이브러리를 생성합니다.

비디오: 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.

Metagenomic 도서관 대규모 화면

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

연구

생물학

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

바이오 - 래드 총 RNA 키트를 사용 Neuroprecursor 셀에서 RNA 추출

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.

인간의 사후 뇌 조직에서 단세포 집단의 고품질 RNA 구하기

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.

Metagenomic 도서관에서 Cellulase 활동 Biomining에 대해 높은 처리량 화면

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.

Barcoded 효모 게놈 라이브러리의 경쟁력 화면

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.

HELA 세포 Transfection 얹는 Gaussia의 루시페라제의 mRNA의 시험 관내 스크립트 작성 및 상한에

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.

염색질 상호 작용을 매핑 및 전사 규정을 이해하는 데 이점 엔드 태그 장면 (ChIA-PET)과 염색질의 상호 작용 분석

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.

사전 mRNA의 기판의 3 &#39;끝 분열 : 휴대폰 무료 시스템을 사용하여 RNA 가공 반응 분석<em&gt; 시험관</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.

저산소증에 따라 단일 세포 수준에서 글로벌 RNA의 합성 분석

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: 셀 형식 관련 Epigenome 프로 파일링

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