이 간행물에 의해 되었습니다 (C. Fritsch) 그랜트 T32ES019851에서에서 자금, R01AG054641, 그리고 멀리 젊은 조사자 부여 (M. Vermulst).

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저자는 공개 없다.

여기, 우리는 꼬마 선 충, 초파리 melanogaster에 Saccharomyces cerevisiae전송 오류 검출을 위한 C-seq 라이브러리의 준비를 위한 최적화 된 프로토콜을 설명합니다. 이 프로토콜에는 대체 기술 뿐만 아니라 기존 프로토콜을 통해 수많은 장점이 있습니다.
지난 15 년 동안 수많은 기자 시스템 개발 되었습니다 luciferase7,8 또?...

게놈 생명의 정확한 생물학 청사진을 제공합니다. 이 청사진을 제대로 구현 하기 좋은 정밀도로 변하게 하는 게놈에 대 한 중요 하다. 그러나, 전사 무료 오류일 가능성이 크다. 예를 들어 RNA polymerases 오류가 시험관에1,2, 하 긴 알려져 있다 그리고 최근에 그들은 오류에서 vivo에서 뿐만 아니라3,,56, 커밋 보였다 DNA에 직면 하는 경우에 특히7,8,,910손상. 함께 찍은, 이러한 관측은 필 사 오류가 발생 지속적으로 모든 살아있는 세포에서 그들은 돌연변이 단백질의 강력한 원천이 될 수 있는 제안 나타냅니다.
Transcriptional mutagenesis 되 나,이 과정에서 두 가지 방법으로 고전 mutagenesis 다릅니다. 첫째, 유전자 변이, 달리 전사 오류 영향을 mitotic 그리고 포스트 mitotic 세포로 DNA 복제에 의존 하지 않는. 전사의 품질에 영향을 주는 메커니즘을 공부 mitotic 그리고 포스트 mitotic 세포의 돌연변이 부하에 대 한 값진 통찰력 제공 따라서. 흥미롭게도, 전사 오류 최근 단백질 집계11,,1213 의 승진에 연루 되어 고 모두 발암10 에 기여할 가설과 항생제 저항 박테리아14의 개발.
둘째, 유전자 변이, 달리 필 사 오류는 자연에 과도. 그들의 임시 존재 하기 때문에 전송 오류 검출 하기 상당히 어려운 특히 도전 이다. 예를 들어 여러 실험실 transcriptional mutagenesis 연구에 대 한 귀중 한 기자가 분석 실험을 고안 했다, 하는 동안 이러한 분석 전사 오류 컨텍스트 및 모델 생물4,15의 한정된 된 수에서 측정 수만 있습니다. 이러한 한계를 극복 하기 위해 많은 연구 자들은 RNA 시퀀싱 기술 (RNA-seq), 이론적으로 모든 종의 transcriptome 걸쳐 기록 전사 오류를 수 있는 게 아닙니다. 그러나, 이러한 연구는 반전 녹음 방송 오류, PCR 증폭 오류 등 자체 시퀀싱의 오류가 자연 도서관 건축 유물에 의해 쉽게 혼동 된다. 예를 들어 역 transcriptases 커밋 약 한 오류 모든 ~ 20000 기지 동안 RNA polymerases (RNAPs) 하나의 실수 모든 300000 기지5,6것으로 예상 된다. 반전 녹음 방송 혼자의 오류율 난쟁이 셀 안에 RNA polymerases의 오류율, 때문에 그것은 전통적인 RNA-Seq 데이터 ( 라이브러리 준비로 인 한 유물에서 진정한 전사 오류를 구별 하는 것이 사실상 불가능 그림 1a).
이 문제를 해결 하려면 우리는 최적화 된 버전 원-시퀀싱 (Cirseq, 또는 C-seq 이제)의 분석 결과5,16을 개발 했다. 이 분석 결과 사용자를 transcriptome5전체 RNA에 전사 오류 및 다른 희소 한 이체를 감지 수 있습니다. 원형-시퀀싱 분석 결과이 분석 결과에 중요 한 단계 주위에 회귀 한다 RNA circularization 때문에이 이름을 수행 합니다. 일단 RNA 대상 circularized는, 그들은 역 같은 RNA 서식 파일의 여러 복사본을 포함 하는 선형 cDNA 분자를 생산 하는 롤링 원 패션에 복사할. 이러한 서식 파일 중 하나에 오류가 있었다면,이 오류 cDNA 분자 내에 포함 된 모든 단일 반복에 것도. 대조적으로, 반전 녹음 방송, PCR 확대, 또는 시퀀싱 오류 무작위로 발생 하는 경향이 하 고 따라서 단 하나 또는 두 개의 반복에 있을 것입니다. 따라서, 각 cDNA 분자에 대 한 합의 시퀀스를 생성 하 여 모든 반복에서 발생 하는 오류에서 임의의 오류를 구별 도서관 건축 유물 효과적으로 진정한 전사 오류 (그림 1b)에서 분리 수 있습니다.
C-seq 분석 결과 정확 하 게 어떤 종의 transcriptome 통해 RNA의 기본 대체, 삽입 및 삭제의 속도 감지를 사용할 수 있는 적절 하 게 사용 하는 경우 (예를 들어 트래버스 및 Ochman17참조). 예, 우리는 C-seq 분석 결과를 사용 단일 기본 해상도5 Saccharomyces cerevisiae, 초파리 melanogaster, 그리고 꼬마 선 충 에서 전사의 오류율의 게놈 넓은 측정을 제공 (관찰 되지 않은). 원래 정확 하 게 RNA 바이러스 인구의 시퀀싱 하는 데 사용, C-seq 분석 결과의 최적화 된 버전이 도서관 건축 유물을 라이브러리 준비 하는 동안 가혹한 조건을 최소화 하기 위해 간소화 되었습니다. 또한, 다양 한 상용 키트를 사용 하 여 분석 결과의 처리량은 크게 개선, 자사의 사용자 뿐만 아니라. 적절 하 게 사용 하는 경우이 분석 결과 정확 하 게 수천 복제, 이전 연구6에 그로 인하여 크게 개선 전송 오류를 검색할 수 있습니다. 전반적으로,이 방법은 transcriptional mutagenesis 연구 하는 강력한 도구 이며 사용자 생물의 넓은 범위에서 녹음의 품질을 제어 하는 메커니즘에 새로운 통찰력을 얻을 수 있습니다.

<table><tbody><tr><td>RiboPure RNA purification kit</td><td>ThermoFisher</td><td>AM1926</td><td>Total RNA purification</td></tr><tr><td>Genelute mRNA purification kit</td><td>Sigma-Aldrich</td><td>MRN70-1KT</td><td>mRNA purification</td></tr><tr><td>Nuclease-free Water</td><td>Ambion</td><td>AM9937</td><td>Elution and dilution</td></tr><tr><td>Ambion RNase III</td><td>ThermoFisher</td><td>AM2290</td><td>RNA fragmentation</td></tr><tr><td>T4 RNA Ligase 1 (ssRNA Ligase)</td><td>New England Biolabs</td><td>M0204S</td><td>RNA circularization</td></tr><tr><td>Ribolock</td><td>ThermoFisher</td><td>EO0381</td><td>RNase inhibitor</td></tr><tr><td>SuperScript III Reverse Transcriptase</td><td>ThermoFisher</td><td>18080044</td><td>Rolling circle reverse transcription</td></tr><tr><td>10 mM dNTP mix</td><td>ThermoFisher</td><td>18427013</td><td>Rolling circle reverse transcription</td></tr><tr><td>Random hexamers (50 ng/&amp;micro;L)</td><td>ThermoFisher</td><td>N8080127</td><td>Rolling circle reverse transcription</td></tr><tr><td>NEB Second Strand Synthesis Module</td><td>New England Biolabs</td><td>E6111S</td><td>Second Strand Synthesis</td></tr><tr><td>NEBNext Ultra DNA Library Prep Kit for Illumina</td><td>New England Biolabs</td><td>E7370S</td><td>cDNA library preparation</td></tr><tr><td>NEB Next index primers</td><td>NEB</td><td>E7335S</td><td>Multiplex PCR primers</td></tr><tr><td>Oligo Clean &amp;amp; Concentrator</td><td>Zymo Research</td><td>D4061</td><td>Clean up of RNA and DNA samples</td></tr><tr><td>DynaMag-2 Magnet</td><td>ThermoFisher</td><td>12321D</td><td>Magnetic bead purification</td></tr><tr><td>AMPure XP beads</td><td>Beckman Coulter</td><td>A63881</td><td>Magnetic bead purification</td></tr><tr><td>Eppendorf 5424 Microcentrifuge</td><td>FisherScientific</td><td>05-403-93</td><td>centrifugation</td></tr><tr><td>INCU-Shaker 10 L</td><td>Benchmark Scientific</td><td>H1010</td><td>Cell culture</td></tr><tr><td>T100 Thermal Cycler</td><td>BIO RAD</td><td>1861096</td><td>Medium to High temperature cycling conditions</td></tr><tr><td>PTC-200 Thermal Cycler</td><td>GMI</td><td>8252-30-0001&amp;nbsp;</td><td>Low temperature cycling conditions</td></tr><tr><td>RNase Away</td><td>Molecular Bioproducts</td><td>700S-11</td><td>Sterilization</td></tr><tr><td>50 mL Centrifuge Tube</td><td>Corning</td><td>430290</td><td>Nuclease-free</td></tr><tr><td>15 mL Centrifuge Tube</td><td>Corning</td><td>430052</td><td>Nuclease-free</td></tr><tr><td>Eppendorf tubes</td><td>USA Scientific</td><td>1615-5500</td><td>Nuclease-free</td></tr><tr><td>4200 Tapestation System</td><td>Agilent</td><td>G2991AA</td><td>Nucleotide analysis instrument for quality control of RNA and single stranded DNA samples</td></tr><tr><td>High Sensitivity RNA Screen Tape</td><td>Agilent</td><td>5067-5579</td><td>Quality control of RNA and single stranded DNA samples</td></tr><tr><td>RNA ScreenTape Sample Buffer</td><td>Agilent</td><td>5067-5577</td><td>Quality control of RNA and single stranded DNA samples</td></tr><tr><td>RNA ScreenTape Ladder</td><td>Agilent</td><td>5067-5578</td><td>Quality control of RNA and single stranded DNA samples</td></tr><tr><td>2100 Bioanalyzer Instrument</td><td>Agilent</td><td>G2939BA</td><td>Double stranded DNA quality control</td></tr><tr><td>High Sensitivity DNA Kit</td><td>Agilent</td><td>5067-4626</td><td>Quality control for double stranded cDNA samples</td></tr><tr><td>Water Bath</td><td>VWR</td><td>462-0244</td><td>Incubation</td></tr><tr><td>NanoDrop 2000/2000C Spectrophotometer</td><td>ThermoFisher</td><td>ND-2000C</td><td>Determination of RNA concentration</td></tr></tbody></table>

genome-wide surveillance of transcription errors in eukaryotic organisms

정확한 전사는 유전 정보의 충실 한 식에 대 한 필요 합니다. 놀랍게도 하지만, 약간 녹음의 품질을 제어 하는 메커니즘에 대 한 알려져 있다. 과학적 지식에이 격차를 채우기 위해 우리는 최근 Saccharomyces cerevisiae, 초파리 melanogaster, 및 꼬마 transcriptome 걸쳐 전사 오류를 감지 하 원-시퀀싱 분석 결과 최적화 선 충. 이 프로토콜은 전사 오류 진 핵 세포에서의 전사의 품질을 제어 하는 메커니즘 전례 없는 자세히 해명 될 수 있도록 지도 하는 강력한 새로운 도구와 연구를 제공할 것입니다.

모든 대규모 병렬 시퀀싱 방법 처럼 각 C-seq 실험 다루기, 큰 데이터 집합을 생성합니다. 처음 사용자를 위해 어려울 수 있습니다; 이러한 데이터 세트를 처리 하기 위해 따라서, 모든 사용자는 실험 전에 경험된 바이오-informatician 문의 하는 것이 좋습니다. 평균, 기대는 사용자 대부분 대규모 병렬 시퀀싱 플랫폼에서 실행 당의 55-70 기가 기지 (Gbases) 약을 생성 합니다. 이 프...

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Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

이 프로토콜 여러 모델 생물에서 녹음의 품질을 모니터링 하는 새로운 도구와 연구를 제공 합니다.

진 핵 생물의 전사 오류의 게놈 넓은 감시

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

genome-wide-surveillance-transcription-errors-eukaryotic

Research

JoVE Journal

Genetics

9.5K 조회수.  Children's Hospital of Philadelphia. 이 프로토콜 여러 모델 생물에서 녹음의 품질을 모니터링 하는 새로운 도구와 연구를 제공 합니다.

비디오: Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here is a powerful experimental approach for analyzing the transcription termination defect under physiological conditions. Like the traditional TRO assay, it relies on the presence of a transcriptionally active polymerase beyond the 3&#8242; end of the gene as an indicator of a transcription termination defect1. It overcomes two major problems encountered with the traditional TRO assay. First, it can detect if the polymerase reading through the termination signal is the one that initiated transcription from the promoter-proximal region, or if it is simply representing a pervasively transcribing polymerase that initiated non-specifically from somewhere in the body or the 3&#8242; end of the gene. Secondly, it can distinguish if the transcriptionally active polymerase signal beyond the terminator region is truly the readthrough sense mRNA transcribing polymerase or a terminator-initiated non-coding anti-sense RNA signal. Briefly, the protocol involves permeabilizing the exponentially growing yeast cells, allowing the transcripts that initiated in vivo to elongate in the presence of the BrUTP nucleotide, purifying BrUTP-labelled RNA by the affinity approach, reverse transcribing the purified nascent RNA and amplifying the cDNA using strand-specific primers flanking the promoter and the terminator regions of the gene2.

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes.

BrUTP 가닥 특정 전사 런타임에 (TRO) 접근 방식을 사용하여 전사의 종료의 분석

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here ...

연구

유전학

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

ChEC-seq와의 단백질 - DNA 상호 작용의 게놈 전체 매핑<em&gt; 사카로 마이 세스 세레 비시 애</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

바인딩, 전사, 번역, 및 회전율 세포 활성화 중 글로벌 이벤트를 표시 하는 녹음 방송 요인의 실시간 분석

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

향상 된 게놈 세포와 유기 체에서 다양 한 Cas9 Ribonucleoprotein 편집

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

포유류 세포에서 미세 Coccal 뉴클레아제사용 염색질 면역침전 분석

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of &#8216;cross-linking and immunoprecipitation&#8217; (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (&#967;CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the &#967;CRAC method and presents recent results obtained with the Nrd1 RBP.

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

χCRAC를 사용하여 높은 시간 분해능에서 생체 내 단백질-RNA 상호 작용 역학 모니터링

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

생화학

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

종양유전자 유래 전사-복제 충돌을 연구하기 위한 근접 결찰 분석

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

암 연구학

Capturing Common Fragile Site Breaks by Native &#947;H2A.X ChIP

Replication stress induced by exposure to extrinsic agents can lead to DNA breaks at common fragile sites, which are regions in the genome known to be prone to structural instability. The &#947;H2A.X chromatin immunoprecipitation (ChIP) assay serves as a powerful tool in genotoxicity studies, as &#947;H2A.X phosphorylation is a well-established marker for DNA double-strand breaks. Traditional &#947;H2A.X ChIP assays, however, are often labor-intensive and involve multiple, time-consuming steps. In this study, we present a simplified yet effective method that combines subcellular fractionation with native ChIP to isolate &#947;H2A.X-associated complexes. This approach is particularly suitable for analyzing &#947;H2A.X-chromatin interactions with enhanced specificity and efficiency. Using subcellular fractionation, chromatin-unbound materials are effectively removed, resulting in a purified chromatin fraction. Subsequent micrococcal nuclease (MNase) digestion under mild conditions allows chromatin fragmentation while preserving physiological interactions between &#947;H2A.X and its associated protein complexes. This preservation is essential for studying native interaction partners involved in DNA damage response pathways. This optimized native ChIP protocol substantially reduces the time and labor associated with conventional &#947;H2A.X ChIP assays. The streamlined procedure not only simplifies the workflow but also yields highly reproducible results, making it particularly advantageous in settings where high-throughput processing of multiple samples is required. This method has broad applicability in studies focused on genome stability, DNA repair, and chromatin biology, where accurate and efficient detection of DNA damage sites is critical. By employing optimized protocols and streamlined steps, this method enables the detection of DNA damage at fragile sites with improved sensitivity and minimal sample handling, making it a valuable tool for studies on genome stability and DNA damage response.

We present a rapid and efficient method to detect common fragile site breaks through native &#947;H2A.X chromatin immunoprecipitation (ChIP). This approach significantly reduces both the time and labor associated with traditional &#947;H2A.X ChIP assays while maintaining high reproducibility and reliability of results.

네이티브 γH2A에 의한 일반적인 취약한 부위 파손 캡처. X 치프

Replication stress induced by exposure to extrinsic agents can lead to DNA breaks at common fragile sites, which are regions in the genome known to be ...

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

세포 배양에서 RNA 합성, 처리 및 부패의 높은 해상도 유전자 발현 프로파일에 대한 새로 전사 RNA 대사 라벨링

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

생물학

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

전기 영동 이동성 분석 (EMSA) 및 Shift 키를 사용하여 기능 비 코딩 유전자 변형에 대한 심사 DNA 화성 강수 분석 (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...