우리는 그녀의 지원과 결실 문제 해결 논의에 참여하기 위해 전체 시그마 생명 과학 미션 대상 아이디 라이브러리 개발팀, 특히 기술을 발견하고 라이센스 조항을 협상에 대한 케빈 Gutshall 및 헤더 Holemon 감사드립니다. 우리는 또한 자유롭게 그것이 복제지고에 큰 cDNA의 배치와 그의 지속성을 준비를 위해 게시되지 않은 결과 제안 및 RxBiosciences의 Qazi 하미드을 공유 킹스 칼리지 런던의 박사 줍 Gäken 감사드립니다. 우리는 cDNA 배열과 데이터 분석을 수행하기 위해 SAFC에서 할머니 린 스콧 Bahr을 인정하고 싶습니다. 목표는 시그마 - 올드 리치 생명 공학 LP의 등록 상표입니다

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+isoform+diversity+can+obscure+detection+of+miRNA-mediated+control+of+translation.">mRNA isoform diversity can obscure detection of miRNA-mediated control of translation.</a> RNA. 17 (6), 1025-1033 (2011).</li></ol>

저자는 제출한 작품에 관심을 가지고 상업적 주체와 재정 관계를 선언합니다.

microRNAs는 억제 mRNA의 번역에 의해 사후 transcriptionally 유전자 발현을 조절하고, 자주, 타겟 mRNA를 ([6]에서 검토) 약화 20-24 염기 RNAs 있습니다. 단일 miRNA는 개발과 환경 신호에 대한 세포의 반응을 제어하기 위해 수백 mRNAs을 조절할 수 있습니다. 식별 및 대상 mRNAs을 확인하면 해당 경로에서 miRNA의 역할과 기능을 결정하는 것이 필수적입니다. 동물에 miRNAs과 타겟 사이트가 완전히 보완하지 않습니다, 때문에, 표적 식별은 간단하지 않습니다. &quot;종자&quot;지역, miRNA의 5&#39; 엔드에서 7을 통해 2 기준으로, 보통의 목표를 보완합니다. 그러나 거기에 씨앗 규칙에 여러 예외이며,베이스 - 페어링 하류는 불완전한 씨앗 경기 보정할 수 있습니다. 컴퓨터 알고리즘의 숫자는 종자 매칭 및 다운 스트림 보상, 대상 구조를 기반 miRNA 목표를 예측하기 위해 개발되었습니다D 위치, 순서 보존, 그리고 실험적으로 검증 대상 ([7]에서 검토)에 대한 관찰되어 여러 가지 다른 매개 변수. 예측이 편리하고 식별 많은 유효 miRNA 목표를 silico에있는 동안 대부분의 유전자는 실험적인 검증 테스트에 실패, 많은 실제 대상이 예측되지 않는 예측했다. 컴퓨터 알고리즘은 이전에 결정된 목표 기능을 기반으로하고 있기 때문에 또한, 그들은 이미 잘 알려져 있습니다 무엇 이탈 표적의 발견을 허용하지 않습니다. 실험 시스템의 숫자는 살아있는 세포의 기능적인 miRNA 목표를 식별하거나 발견하는 데 성공적으로 사용되었습니다. 이러한 글로벌 심사 방법은 microarrays와 RNA 시퀀싱, RNA 공동 immunoprecipitation (RIP) 및 세포 배양에서 아미노산과 안정 동위 원소 라벨링 (SILAC), proteomic 방법 ([7]에서 검토)가 있습니다. 각 방법은 장점과 단점이 있습니다. 타겟팅 miRNA는 종종 mRNA를 destabilizes과 열화, 분실 O로 연결하기 때문에miRNA는 각각 모방하거나 억제제를 도입 후 mRN​​A 수준에서 R 게인은 miRNA 목표를 식별할 수 있습니다. mRNA의이 손실이나 이득은 쉽게 microarray 또는 크게 시퀀싱에 의해 감지된다. mRNA 검출 방법은 간단하고 단백질 검출 방법보다 더 민감한 반면, mRNA 탐지가 타락되지 않은 miRNA 목표를 놓칠 것이다. SILAC에서 이들과 함께 RNA 검출에서 miRNA 목표 결과를 비교 Bartell 연구소의 최근 보고서 [8] 또는 ribosome 파일링 [9] 포유 동물 miRNAs는 주로 타겟 mRNA 수준을 줄임으로써 행동을 나타냅니다. 그러나 개별 miRNA 목표와 협력 많은 다른 연구진이 단백질의 변화하지만 mRNA 수준에는 변화를 감지했습니다. 노 감지 mRNA의 손실과 translational 규제하는 것으로 보이는 보고서는 다음과 같습니다 미르-10B HOXD10에 [10] p27kip1에서 miR221/222 [11] Pd​​cd4에서 미르-21 [12] 미르-126 p85β에 [13] , SIRT1의 미르-34 [14] PTEN [15] 미르-302d Arid4b에 [16] 미르-200c JAG1에 [17], 그리고 미르-299, 297, 567,에, 미르-21VEGFA에 ND 609 [18]. 또한, 클랜 외 [19] 최근 가주-7 대상 사이트를 포함하는 각각의 mRNA의 isoforms이 선택적으로 발견되었을 때 가주-7에 의해 translational 규제에만 발견되었다고 보도했다. 모든 mRNA의 isoforms 하나의 mRNA로 감지되면 그것은, -로 - 많은 microarray 및 시퀀스 분석으로 얻은 후자는 최소한 어떤 경우에는, translational 규제가​​ 복합 프로파일에 간과할 수있다,하는 것이 좋습니다. argonaute (보통 ago2) 또는 다른 관련된 단백질 (RNA immunoprecipitation, 또는 RIP)를 통해 miRNA-mRNA의 단지의 공동 immunoprecipitation에 관계없이 규제 메커니즘의 miRNA 목표를 분리합니다. 또한, RIP는 상호 작용을 내생, 그리고 아마도 생물학적으로 관련성이 감지합니다. 그러나, 그것은 모든 miRNA-mRNA의 상호 작용 기능이며, RIP 만 transiently 연결하거나 급속하게 저하에있다는 mRNA 목표를 놓칠 것인 지 알 수 없습니다. 또한, 특정 miRNA-mRNA 파트너는 bioinformati에게 유추되어야합니다모든 miRNA-mRNA 쌍 함께 공동 시켰던 캘리 때문입니다. 마지막으로, SILAC 직접 miRNA 조절, 단백질 자체의 최종 제품을 식별하지만, 구별이므로 희귀 단백질 및 단백질 수준의 작은 배 변화를 그리워합니다. 현재 miRNA 목표 식별 방법과 한계에 비추어, 우리는 다른 메커니즘에 의해 기능적인 miRNA 목표를 식별하기위한 추가적인 글로벌 분석이 필요했다 느꼈다. 이러한 필요성을 충족하기 위해 줍 Gaken와 킹스 칼리지 런던의 Azim Mohamedali에 의해 발명 기술을 허가. 그들의 발명은 이중 선택 융합 단백질이며, 특히, thymidine 키나제-zeocin 융합, 그 3&#39;UTR의 잠재적인 miRNA 목표 시퀀스의 cDNA 라이브러리에 의해 규제. 그림 2에서 그림과 같이 안정적으로 TKzeo-cDNA 구조와 함께 표현 transfected 세포는 zeocin으로 선택할 수 있습니다. 관심 miRNA을 표현하면, 그 miRNA의 목표 이래 선택할 수 있습니다일 ganciclovir와 같은 그림 3 그림. Ganciclovir는 모든 세포의 관심 miRNA에 대한 목표를 부족한 TKzeo-cDNA 표출하는 thymidine의 키나제를 표현하는 세포를 죽이는. 타겟 cDNA는 측면 cDNA와 PCR 제품은 목표를 식별하기 위해 순서가 수 ganciclovir 사용 primers를 생존 세포에서 DNA의 PCR 증폭에 의해 격리 수 있습니다. 선교 대상 아이디 도서관 - 저희 기능성 인간 miRNA 목표의 글로벌 식별 및 검색을위한 새로운 도구에 국왕의 대학 기술을 개발했습니다. 대상 아이디 라이브러리를 통해 사용자는 포유 동물 세포 배양 transfection과 약물 선택 일련의 단계에 의해 miRNA 목표를 분리할 수 있습니다. 도서관은 포괄적이며, 인간 유전자의 66-79%가 포함되어 있습니다. 초기 결과는 모두 이전에 발견뿐만 아니라 새로운 목표는 선교 대상 ID를 도서관에서 격리 수 있음을 나타냅니다. 프로토콜은 몇 가지 길이 있지만포유 동물 세포 배양, transfection, 약물 선택, PCR 및 시퀀싱 - - 타겟 ID 라이브러리의 사용은 표준 분자 실험 기법을 필요로하므로, 대부분의 생물학 자의 수단 내에서 잘해야합니다. 우리는 충분한주의가 문화 환경을 최적화, 적절한 실험 설계에 납부하고, 가장 중요한 목표 아이디 라이브러리 성공을 보장하는 선택 단계 (죽이기-커브)에 대한 최적의 약물 수준을 결정하기 위해 각각의 새로운 세포 유형을 미리 테스트하는 것이 좋습니다. 모든 miRNA 목표 식별 방법과 마찬가지로, 그것은 높은 대상 아이디 도서관을 분석해 식별 대상이 같은 microarray, qRT-PCR, 기자 분석 (즉, 루시페라제) 또는 서양 분석과 같은 두 번째 방법과 함께 확인하는 것이 좋습니다.

<table border="1" ><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> Jumpstart를 REDTaq ReadyMix </td><td> 시그마 - 올드 리치 </td><td> P0982 </td><td></td></tr><tr><td> Ganciclovir </td><td> 시그마 - 올드 리치 </td><td> G2536 </td><td></td></tr><tr><td> G418 </td><td> 시그마 - 올드 리치 </td><td> G8168 </td><td></td></tr><tr><td> Puromycin </td><td> 시그마 - 올드 리치 </td><td> P9620 </td><td></td></tr><tr><td> Topo TA </td><td> Invitrogen </td><td> KNM4500-01 </td><td></td></tr><tr><td> 포유류의 게놈 DNA를 Miniprep 키트 GenElute </td><td> 시그마 - 올드 리치 </td><td> G1N10 </td><td></td></tr><tr><td> 셀 특정 Nucleofection의 K그것 </td><td> Lonza </td><td></td><td></td></tr><tr><td> Nucleofector </td><td> Lonza </td><td> AAD-1001 </td><td></td></tr><tr><td> Zeocin </td><td> Invitrogen </td><td> <a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=R25001">R250-01</a> </td><td></td></tr><tr><td height="1"> 대상 ID를 도서관 </td><td> 시그마 - 올드 리치 </td><td> MREH01 </td><td></td></tr></tbody></table>

genome-wide screen for mirna targets using the mission target id library

대상 아이디 라이브러리는 microRNA의 발견 및 식별 (miRNA) 목표를 지원하도록 설계되었습니다. 대상 아이디 도서관은 이중 선택 융합 단백질, thymidine 키나제-zeocin (TKzeo)에서 3&#39;UTR의 하류에 복제된 플라스미드 기반의 게놈 전체 cDNA 라이브러리입니다. 선택의 첫 번째 라운드는 안정 transformants에 대한 관심이 miRNA의 도입에 따라, 그리고 마지막으로, miRNA의 목표를 포함하는 cDNAs을위한 선택입니다. 선택된 cDNAs은 시퀀싱 (대상 아이디 라이브러리 워크플로우 및 자세한 내용은 그림 1-3 참조)에 의해 식별됩니다. 인간의 transcriptome의 광범위한 적용 범위를 보장하기 위해 대상 ID가 도서관 cDNAs은 여러 사람의 조직 및 세포 라인에서 준비한 총 RNA의 수영장을 사용하여 oligo-DT의 마중물을 통해 생성되었습니다. 1.2 킬로바이트의 평균 크기, 0.5에서 4 KB로 cDNA 범위를 결과 및 (플라스미드지도 그림 4 참조) p3TKzeo 이중 선택 플라스미드로 복제되었다. 리로 표현 유전자 표적brary는 시그마 - 올드 리치 웹 페이지에서 찾을 수 있습니다. Illumina 시퀀싱 (표 3)의 결과가 도서관 UCSC RefGene에서 21,518 고유한 유전자 16,922 (79 %), 또는 10 또는 읽습 이상 (66 %)로 14,000의 유전자를 포함 것을 보여줍니다.

Watch this Scientific Journal Video about Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library at JoVE.com

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

대상 아이디 라이브러리 miRNA 목표를 식별하는 데 사용 복제된 cDNA의 플라스미드 기반의 게놈 전체 모음입니다. 여기는 이용 및 응용 프로그램을 보여줍니다.

선교 대상 아이디 라이브러리를 사용하여 miRNA 목표를위한 게놈 - 와이드 스크린

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

genome-wide-screen-for-mirna-targets-using-mission-target-id

Nanyang Technological University

Research

JoVE Journal

Biology

17.5K 조회수.  Sigma-Aldrich. 대상 아이디 라이브러리 miRNA 목표를 식별하는 데 사용 복제된 cDNA의 플라스미드 기반의 게놈 전체 모음입니다. 여기는 이용 및 응용 프로그램을 보여줍니다.

비디오: Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

Microarrays을 사용하여 tRNA의 Aminoacylation (충전) 레벨의 게놈 - 와이드 분석

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

연구

생물학

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

이소형 특정 유전자 목표를 식별 칩을 사용하여 게놈 차원의 분석

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

임무 LentiPlex는 포유 동물 세포에서 shRNA 도서관 검사를 풀링

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

의 정량 및 자동화된 고속 처리량 게놈 전체의 RNAi 화면 C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of protein-bound DNA and aligning the short reads to a reference genome. Enriched regions are revealed as peaks, which often differ dramatically in shape, depending on the target protein1. For example, transcription factors often bind in a site- and sequence-specific manner and tend to produce punctate peaks, while histone modifications are more pervasive and are characterized by broad, diffuse islands of enrichment2. Reliably identifying these regions was the focus of our work.
Algorithms for analyzing ChIPseq data have employed various methodologies, from heuristics3-5 to more rigorous statistical models, e.g. Hidden Markov Models (HMMs)6-8. We sought a solution that minimized the necessity for difficult-to-define, ad hoc parameters that often compromise resolution and lessen the intuitive usability of the tool. With respect to HMM-based methods, we aimed to curtail parameter estimation procedures and simple, finite state classifications that are often utilized.
Additionally, conventional ChIPseq data analysis involves categorization of the expected read density profiles as either punctate or diffuse followed by subsequent application of the appropriate tool. We further aimed to replace the need for these two distinct models with a single, more versatile model, which can capably address the entire spectrum of data types.
To meet these objectives, we first constructed a statistical framework that naturally modeled ChIPseq data structures using a cutting edge advance in HMMs9, which utilizes only explicit formulas-an innovation crucial to its performance advantages. More sophisticated then heuristic models, our HMM accommodates infinite hidden states through a Bayesian model. We applied it to identifying reasonable change points in read density, which further define segments of enrichment. Our analysis revealed how our Bayesian Change Point (BCP) algorithm had a reduced computational complexity-evidenced by an abridged run time and memory footprint. The BCP algorithm was successfully applied to both punctate peak and diffuse island identification with robust accuracy and limited user-defined parameters. This illustrated both its versatility and ease of use. Consequently, we believe it can be implemented readily across broad ranges of data types and end users in a manner that is easily compared and contrasted, making it a great tool for ChIPseq data analysis that can aid in collaboration and corroboration between research groups. Here, we demonstrate the application of BCP to existing transcription factor10,11 and epigenetic data12 to illustrate its usefulness.

Our Bayesian Change Point (BCP) algorithm builds on state-of-the-art advances in modeling change-points via Hidden Markov Models and applies them to chromatin immunoprecipitation sequencing (ChIPseq) data analysis. BCP performs well in both broad and punctate data types, but excels in accurately identifying robust, reproducible islands of diffuse histone enrichment.

다양한 ChIPseq 데이터 유형의 게놈 차원의 분석을위한 소설 베이지안 변경 - 포인트 알고리즘

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

게놈 전체 유전자 삭제할에<em&gt; 연쇄상 구균 sanguinis</em&gt; 높은 처리량 PCR에 의한

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

게놈 전체 규모에 Polysom​​e 분별과 포유류 Translatomes 분석

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

3&#39;LIFE의 분석을 사용하여 높은 처리량 miRNA의 표적의 탐지

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

COSMIC와 세포의 약물-DNA 상호 작용의 게놈 넓은 매핑 (작은 분자의 가교는 염색질을 분리)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

진핵 세포에서 염색체 외 원형 DNA의 게놈 차원의 정화

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...