Biz ona destek ve sorun giderme verimli tartışmalara katılım için tüm Sigma Hayat Bilgisi Misyon Hedef Kimliği Kütüphane geliştirme ekibi, özellikle teknoloji bulmak ve lisans koşullarını müzakere Kevin Gutshall ve Heather Holemon teşekkür ederiz. Biz de özgürce klonlanmış almak büyük bir cDNA parti ve onun kalıcılığını hazırlamak için yayımlanmamış sonuçlar ve öneriler ve RxBiosciences ve Gazi Hamid paylaşımı için King College London Dr Joop Gäken teşekkür ederim. Biz cDNA dizileri ve veri analizi gerçekleştirmek için SAFC gelen Nan Lin ve Scott Bahr kabul etmek istiyorum. MİSYON Sigma-Aldrich Biyoteknoloji LP tescilli markasıdır

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Biol. 17 (10), 1169-1174 (2010).</li><li>Baek, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+microRNAs+on+protein+output.">The impact of microRNAs on protein output.</a> Nature. 455 (7209), 71-71 (2008).</li><li>Guo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+microRNAs+predominantly+act+to+decrease+target+mRNA+levels.">Mammalian microRNAs predominantly act to decrease target mRNA levels.</a> Nature. 466 (3708), 835-840 (2010).</li><li>Ma, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+invasion+and+metastasis+initiated+my+microRNA-10b+in+breast+cancer.">Tumour invasion and metastasis initiated my microRNA-10b in breast cancer.</a> Nature. 449 (7163), 682-688 (2007).</li><li>Visone, R., 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=MicroRNAs+miR-221+and+miR-222,+both+overexpressed+in+human+thyroid+papillary+carcinomas,+regulate+p27Kip1+protein+levels+and+cell+cycle.">MicroRNAs miR-221 and miR-222, both overexpressed in human thyroid papillary carcinomas, regulate p27Kip1 protein levels and cell cycle.</a> Endocrine-Related Cancer. 14 (3), 791-798 (2007).</li><li>Lu, Z., 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=MicroRNA-21+promotes+cell+transformation+by+targeting+the+programmed+cell+death+4+gene.">MicroRNA-21 promotes cell transformation by targeting the programmed cell death 4 gene.</a> Oncogene. 27 (31), 4373-4379 (2008).</li><li>Guo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+noncoding+RNA,+miR-126,+suppresses+the+growth+of+neoplastic+cells+by+targeting+phosphatidylinositol+3-kinase+signaling+and+is+frequently+lost+in+colon+cancers.">The noncoding RNA, miR-126, suppresses the growth of neoplastic cells by targeting phosphatidylinositol 3-kinase signaling and is frequently lost in colon cancers.</a> Genes Chrom. Cancer. 47 (11), 939-946 (2008).</li><li>Yamakuchi, M., 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=miR-34a+repression+of+SIRT1+regulates+apoptosis.">miR-34a repression of SIRT1 regulates apoptosis.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (36), 13421-13426 (2008).</li><li>Wickramasinghe, N. S., 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=Estradiol+downregulates+miR-21+expression+and+increases+miR-21+target+gene+expression+in+MCF-7+breast+cancer+cells.">Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells.</a> Nucleic Acid Res. 37 (8), 2584-2595 .</li><li>Ciaudo, C., 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=Highly+dynamic+and+sex-specific+expression+of+microRNAs+during+early+ES+cell+differentiation.">Highly dynamic and sex-specific expression of microRNAs during early ES cell differentiation.</a> PLoS Genetics. 5, e1000620 (2009).</li><li>Vallejo, D. M., 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=Targeting+Notch+signaling+by+the+conserved+miR-8/200+microRNA+family+in+development+and+cancer+cells.">Targeting Notch signaling by the conserved miR-8/200 microRNA family in development and cancer cells.</a> EMBO J. 30 (4), 756-769 (2011).</li><li>Jafarifar, F., 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=Repression+of+VEGFA+by+CA-rich+element-binding+microRNAs+is+modulated+by+hnRNAP.">Repression of VEGFA by CA-rich element-binding microRNAs is modulated by hnRNAP.</a> L. EMBO J. 30 (7), 1324-1334 (2011).</li><li>Clancy, J. 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>

Yazarlar gönderilen eser bir ilgi ticari kuruluşlar ile mali ilişkileri beyan ederim.

microRNA inhibe mRNA çeviri post-transkripsiyonel gen ekspresyonu düzenleyen ve sık sık, hedeflenen mRNA ([6] Yorumlar) istikrarsızlaştırma 20-24 nükleotid RNA&#39;lar vardır. Tek miRNA gelişimsel ve çevresel sinyallere bir hücre tepkisi kontrol etmek için birçok yüz mRNA&#39;ların düzenleyebilir. Belirlenmesi ve hedef mRNA doğrulayarak bu yolları bir miRNA rolü ve fonksiyonu belirlemede önemlidir. Hayvanlarda, miRNA&#39;lar ve hedef siteler tamamen tamamlayıcı değildir, çünkü Ancak, hedef tespit kolay değildir. &quot;Tohum&quot; bölge, miRNA ve 5&#39;-ucundan 2 ile 7 arasındaki dayandırır, genelde hedeflerini tamamlayıcısı oluyor. Ancak, tohum kuralın pek çok istisnası vardır ve taban-eşleştirme aşağı bir kusurlu tohum maçı telafi edebilir. Bilgisayar algoritmaları Bir çok tohum eşleştirme ve mansap tazminat, hedef yapısına dayalı miRNA hedefleri tahmin etmek geliştirilmiştird konumu, sıra korunması ve deneysel olarak doğrulanmış hedefler ([7] Yorumlar) için gözlenmiştir diğer çeşitli parametreler. Öngörüleri uygun olan ve tanımlamaya pek çok geçerli miRNA hedefleri do silico iken, en genler deneysel doğrulama testleri başarısız ve birçok gerçek hedefleri tahmin edilmiyor öngördü. Bilgisayar algoritmaları önceden belirlenmiş hedef özelliklere dayalı Dahası, bunlar zaten bilinen sapan hedeflerin keşfi izin vermez. Deneysel sistem bir dizi canlı hücrelerin işlevsel miRNA hedefleri belirlemek veya keşfetmek için başarıyla kullanılmaktadır. Bu küresel tarama yöntemleri mikroarrayler ve RNA sıralama, RNA co-immünopresipitasyon (RIP), ve Hücre kültüründe Amino asitler ile Kararlı İzotop Etiketleme (SILAC), bir proteomik yöntemi ([7] &#39;de gözden) içerir. Her yöntemin avantajları ve dezavantajları vardır. Hedefleme miRNA genellikle mRNA oynattığını ve onun bozulması, kaybolması o yol açar yanaBir miRNA sırasıyla, taklit ya da inhibitör tanıttıktan sonra mRNA düzeyinde r kazanç, miRNA hedefler belirleyebilir. MRNA Bu kaybı veya kazancı kolaylıkla mikroarray veya derin sekanslama tarafından tespit edilir. MRNA algılama yöntemleri basit ve protein tespit yöntemleri daha duyarlı iken, mRNA algılama bozulmuş olmayan herhangi bir miRNA hedefleri özleyeceğim. SILAC gelenler RNA algılamayı miRNA hedef sonuçlarını karşılaştırarak Bartell laboratuvar gelen son haberler [8] veya ribozom profilleme [9] memeli miRNA&#39;lar ağırlıklı hedef mRNA düzeylerini azaltarak etki gösterir. Ancak, bireysel miRNA hedefleri ile çalışan diğer birçok laboratuvarlarında protein değişir ama mRNA düzeyinde herhangi bir değişiklik tespit ettik. Saptanabilir mRNA kaybı ile translasyonel düzenlemesi için ne görünür Raporları şunlardır: miR-10b HOXD10 [10] p27kip1 üzerine, miR221/222 [11] Pdcd4 üzerine, miR-21 [12], miR-126 p85β üzerinde [13] , SIRT1 üzerine miR-34 [14] PTEN [15], miR-302d Arid4b [16], miR-200C JAG1 [17], ve miR-299, 297, 567, bir üzerine, miR-21VEGFA üzerine nd 609 [18]. Ayrıca, Clancy ve ark [19] yakın let-7 hedef sitesi içeren bireysel mRNA izoformları seçici tespit edildiğinde let-7 tarafından translasyonal yönetmelik sadece tespit edildiğini bildirdi. Tüm mRNA izoformları bir mRNA olarak tespit edildiğinde yani, - - kadar mikroarray ve sekans analizi ile elde edilen ikinci en azından bazı durumlarda, translasyon düzenlemesi, bir kompozit profili göz ardı edilebilir, göstermektedir. Argonaute (genellikle ago2) veya başka bir ilişkili protein (RNA immünopresipitasyon veya RIP) ile miRNA-mRNA komplekslerinin Co-immünopresipitasyon bağımsız regülasyon mekanizması miRNA hedefleri izole olacaktır. Ayrıca, RIP, etkileşimleri endojen ve muhtemelen biyolojik ilgili algılar. Ancak, tüm miRNA-mRNA etkileşimleri fonksiyonel olup, RIP, yalnızca geçici ilişkilendirmek veya hızla bozulmuş olan herhangi bir mRNA hedefleri kaçıracağı olup olmadığı bilinmemektedir. Ayrıca, özel miRNA-mRNA ortakları bioinformati varılabilir gerekirTüm miRNA-mRNA çiftleri birlikte co-çöktürülmüş Cally çünkü. Son olarak, SILAC doğrudan miRNA düzenlemesi, protein kendisinin son ürün tanımlar, fakat duyarsız olduğunu ve bu nedenle nadir proteinler ve protein düzeyleri küçük değişiklikler kat bahsi. Mevcut miRNA hedef belirleme yöntemleri ile sınırlamalar ışığında, biz alternatif bir mekanizma ile fonksiyonel miRNA hedefleri belirlemek için ek bir küresel analiz ihtiyaç hissettim. Bu ihtiyacı karşılamak üzere, Joop Gaken ve King s College London Azim Mohamedali tarafından icat edilmiş bir teknoloji lisanslı. Bunların buluş, bir çift seçimi füzyon proteini olup, özellikle, bir timidin kinaz-zeocin füzyon, onun 3&#39;UTR potansiyel miRNA hedef dizilerinin bir cDNA kütüphanesi düzenlenir. Şekil 2 de gösterildiği gibi, stabil bir şekilde TKzeo-yapıları cDNA ile transfekte edilmiş ve eksprese eden hücreler, zeocin seçilebilir. Bir ilgi miRNA ifade ettikten sonra, bu miRNA hedefleri wi seçilebilirth gansiklovir, Şekil 3 &#39;de gösterilmiştir. Gansiklovir, herhangi bir hücre ilgi miRNA için bir hedef yoksun TKzeo-cDNA ifade edilir timidin kinaz, Salgılayan hücrelerin öldürür. Hedeflenen cDNA kanadını cDNA ve PCR ürünleri hedefleri tespit etmek için diziliş edilebilir gansiklovir kullanılarak hayatta primerler hücrelerinden DNA&#39;nın PCR amplifikasyonu ile izole edilebilir. MİSYON Hedef Kimliği Kütüphane - Fonksiyonel insan miRNA hedeflerinin küresel kimlik ve keşif için yeni bir araç haline King &#39;s College teknolojisi geliştirdi. Hedef Kimliği Kütüphane ile kullanıcılar, memeli hücre kültürü transfeksiyon ve ilaç seçimi bir dizi adım tarafından miRNA hedefleri ayırabilirsiniz. Kütüphane kapsamlı olduğunu ve insan genlerinin% 66-79 içerir. İlk sonuçlar, hem daha önce keşfedilen yanı sıra yeni hedefler MİSYON Hedef Kimliği Kütüphane izole edilebileceğini göstermektedir. Protokolü, bazı uzunluğunun olmasına rağmenmemeli hücre kültürü, transfeksiyon, ilaç seçimi, PCR ve sıralama - - Hedef Kimliği Kütüphane kullanımı standart moleküler laboratuvar teknikleri gerektirir ve bu yüzden biyologların çoğu, araçlarının içinde de olmalıdır. Biz yeterince ilgi kültür koşullarının optimize, uygun deneysel tasarım için para ve en önemlisi, Hedef Kimliği Kütüphanesi ile başarı sağlamak için seçim adımları (kill-eğrileri) için optimum ilaç düzeylerini belirlemek için her yeni hücre tipi ön test edilmesini önerme. Tüm miRNA hedef tanımlama yöntemleri olduğu gibi, son derece Hedef Kimliği Kütüphane eleme tarafından belirlenen hedefler gibi mikroarray, QRT-PCR, reporter assay (yani, lusiferaz) veya Batı analizi gibi ikinci bir yöntem ile doğrulanması önerilir.

<table border="1" ><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlar </td></tr><tr><td> JumpStart REDTaq Readymix </td><td> Sigma-Aldrich </td><td> P0982 </td><td></td></tr><tr><td> Gansiklovir </td><td> Sigma-Aldrich </td><td> G2536 </td><td></td></tr><tr><td> G418 </td><td> Sigma-Aldrich </td><td> G8168 </td><td></td></tr><tr><td> Puromycin </td><td> Sigma-Aldrich </td><td> P9620 </td><td></td></tr><tr><td> Topo TA </td><td> Invitrogen </td><td> KNM4500-01 </td><td></td></tr><tr><td> Memeli Genomik DNA Miniprep Takımı GenElute </td><td> Sigma-Aldrich </td><td> G1N10 </td><td></td></tr><tr><td> Hücre Özgül Nucleofection ko </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"> Hedef Kimliği Kütüphane </td><td> Sigma-Aldrich </td><td> MREH01 </td><td></td></tr></tbody></table>

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

Hedef Kimliği Kütüphane mikroRNA keşif ve kimlik (miRNA) hedeflerine yardımcı olmak için tasarlanmıştır. Hedef Kimliği Kütüphane çift seçimi füzyon proteini, timidin kinaz-zeocin (TKzeo) den 3&#39;UTR aşağı klonlanmış bir plazmid tabanlı, genom cDNA kütüphanesi olduğunu. Seçimin ilk turunda, istikrarlı transformantlar için bir ilgi miRNA tanıtımı ile takip, ve nihayet, miRNA hedefi içeren cDNAlarının için seçimi olduğunu. Seçilen cDNAlarının sıralama (Hedef Kimliği Kütüphane İş Akışı ve ayrıntılar için bkz. Şekil 1-3) ile tanımlanır. Insan transcriptome geniş kapsama sağlamak için, Hedef Kimliği Kütüphane cDNAlarının birden fazla insan doku ve hücre hatları hazırlanan toplam RNA havuzu kullanarak oligo-dT astar ile oluşturulmuştur. 1.2 kb arasında bir ortalama boyutu olan, 0.5 ile 4 kb için cDNA aralığı çıkan ve (plazmid haritası için bakınız Şekil 4) p3TKzeo ikili seçme plasmid içine klonlandı. Li temsil gen hedefleribrary Sigma-Aldrich web sayfasında bulunabilir. Illumina sekanslama (Tablo 3), elde edilen sonuçlar, kütüphanesi UCSC RefGene içinde 21.518 eşsiz genlerin 16.922 (% 79), ya da 10 ya da daha fazla okur (% 66) ile 14.000 genler dahil olduğunu göstermektedir.

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

Hedef Kimliği Kütüphane miRNA hedefleri belirlemek için kullanılan klonlanmış cDNA bir plazmid tabanlı, genom koleksiyondur. Burada kullanımı ve uygulama göstermektedir.

MİSYON Hedef Kimliği Kütüphane Kullanımı miRNA Hedefler için genom Ekran

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

Research

JoVE Journal

Biology

17.5K Görüntüleme.  Sigma-Aldrich. Hedef Kimliği Kütüphane miRNA hedefleri belirlemek için kullanılan klonlanmış cDNA bir plazmid tabanlı, genom koleksiyondur. Burada kullanımı ve uygulama göstermektedir.

Video: 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.

Mikroarray&#39;ler kullanarak tRNA Aminoacylation (Şarj) Seviyeler genom Analizi

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

Biyoloji

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.

Izoform özel Gen Hedefler Belirlemek için ChIP kullanarak genom analizi

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.

MİSYON LentiPlex Memeli Hücreleri shRNA Kütüphane Tarama Pooled

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.

Nicel ve Otomatik Yüksek throughput Genom çapında RNAi Ekranlar 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.

Çeşitli ChIPseq Veri Türleri Genom çapında Analizi İçin Yeni Bir Bayesian değiştir nokta Algoritması

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.

Genom çapında Gen Delesyonlar içinde<em&gt; Streptococcus sanguinis</em&gt; Yüksek Verimli PCR ile

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

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.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

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;LIFE Test kullanma Yüksek throughput miRNA Hedefleri tespiti

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).

COSMIC ile Hücrelerde İlaç-DNA Etkileşimlerinin genom Haritalama (Küçük moleküllerinin çapraz bağlanmasını Kromatin yalıtmak için)

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.

Ökaryotik Hücrelerde gelen ekstrakromozomal dairesel DNA genom Arıtma

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

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

Genom kapsamındaki bir tanımlama ve ifade Meta-Analizi ATL E3 Ubikuitin ligaz gen ailesinin asma için kapsamlı iş akışı

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...