The authors would like to thank members of the Yi laboratory especially Zhaojie Zhang for fruitful discussions regarding linker design and ligation efficiencies, as well as the American Cancer Society for supporting this work through a postdoctoral fellowship (#125209) to J.E.L. Research reported in this publication was also supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01AR059697 (to R.Y.) and a research grant from the Linda Crnic Institute for Down Syndrome. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Not: Bu, tüm işlem boyunca RNase içermeyen koşulları sağlamak için çok önemlidir. 3 &#39;Bağlayıcının 1. Adenylation <ol><li> Nükleaz içermeyen H2O içinde 100 uM 3 ligasyon reaksiyonları için tasarlanmış DNA oligonükleotid seyreltin Not: oligonükleotidi bir 5 &#39;fosfat grubu, 5 bir randomize dinükleotid&#39; ucuna ve 3 &#39;dideoxycytosine olmalıdır. 5 PCP 10 &#39;fosfat grubu etkili 5 için Mth enzimin bir gerekliliktir&#39;. Fosfat doğrudan modifikasyonu ile bir polinükleotid kinaz ile oligonükleotidin ön-muamele ile ilave ya da istenebilir. 5 &#39;ucunda, randomize dinükleotit hangi belirli bir son uç diğerleri üzerinde 6 tepkileri birleştirilmesi için bir RNA ligaz sergiler sekansı tercih en aza indirmek için önemlidir. 3, oligonükleotid t &#39;ucuna dideoxycytosine 3 bir blokaj elemanının yerleştirir&#39;O sonraki bağlama adımları sırasında bağlayıcı-bağlayıcı konkatamerler oluşumunu inhibe eden ve aynı zamanda bağlama reaksiyonu, 11 3 &#39;3&#39;ün ucunda oluşturulan miRNA-Bağlayıcı molekülün ucunu&#39; için blok teşkil eder. Aşağıdaki gibi tarafsız miR-Dizi yöntemi için seçilen oligonükleotid linker dizisi olduğu için: 5&#39;PO 4 NNTGGAATTCTCGGGTGCCAAGG-dideoxyC3. &#39; </li><li> 200 pmol 3 &#39;oligonükleotidi, 4 ul 10x için 5&#39; DNA adenilasyon tampon maddesi, 4 ul 1 mM ATP, 4 ul Mde RNA ligaz, nükleaz içermeyen H2O 40 ul&#39;lik nihai bir hacme kadar: Aşağıdaki adenilasyon reaksiyon bir araya getirin. </li><li> 1 saat boyunca 65 ° C&#39;de reaksiyon inkübe edin. 5 dakika boyunca 85 ° C&#39;ye ısıtılması yolu ile reaksiyonu bitirin. </li><li> Kalıntı ATP kaldırmak ve gelecekteki reaksiyonlarda beklenmedik ligasyonu önlemek için% 100 2.5 hacim etanol ve 10 M amonyum asetat 1/3 hacme ilavesiyle adenylated bağlayıcı çökeltilmesi. <ol><li> Kısaca, al karıştırıncohol, tuz ve 20 dakika boyunca 4 ° C&#39;de bir mikrosantrifüj içinde tam hızda eğirme homojenlik oligo çözeltisi (~ 15,000 xg). Adenylated oligo 50-100 uM vermek üzere nükleaz içermeyen H2O uygun hacimde% 70 etanol, kuru hava ve tekrar süspansiyon pelet yıkayın. </li></ol></li><li> % 18 poliakrilamidjeli çalıştırarak adenilasyon onaylayın. <ol><li> Birlikte, aşağıdakileri karıştırarak jel hazırlayın: 6.3 g üre,% 40 akrilamid 6.75 mi, ve 10x TBE 1.5 mi. H 2 O 15 ml ilave edilir. </li><li> Üre tamamen çözülene kadar karıştırın ve amonyum persülfat (BP), ve 15 ul tetramethlyethylenediamine (TEMED) (ağ / hac), 150 ul% 10 ekleyin. </li><li> Jel 1x TBE oda sıcaklığında tampon maddesi ile jel genişliğinin 24 V / cm &#39;de 30 dakika boyunca ön-run kurduğunda. Tamponu ile çalışan 30 dk kadar bir kez yıkayın kuyular. </li><li> Ile adenylated oligonükleotitlerin ve 2x denatüre RNA yükleyici tampon eşit hacimde (% 95 (h / h) formamid 5 pmol karıştırın% 0.02 SDS,% 0.02 bromofenol mavisi (ağırlık / hacim),% 0.01 (ağırlık / hacim) (w / v) Ksilen siyanol, 1.0 mM EDTA) içinde, ısı kısa bir süre için 75 ° C ve buz üzerinde soğutuldu oturmaktadır. Jelin kuyuya, örneğin tamamına koyun. Aynı zamanda, bir sigara adenylated kontrol benzer bir miktar ve düşük molekül ağırlıklı boyutu standartlarına uygun bulunmaktadır. </li><li> Bromofenol mavi jel tabanına yol ¾&#39;ünü kadar 24 V / cm jel üzerinde çalıştırıldı. 1x TBE 1x SYBR-altın çözeltisi ile cihaz, post-leke jel sökmeye ve UV-transilluminator&#39;de kutusu (Şekil 2) görselleştirmek. Not: girdi RNA örnekleri ile karşılaştırıldığında Jel &quot;, herhangi bir giriş RNA &#39;olarak kontrol PCR ürününün yüksek miktarda elde edilir, yukarıda tarif edilene benzer bir şekilde adenylated oligo temizler. </li></ol></li></ol> 2. Bağlayıcı Ligasyon miRNA 3 &#39;ucuna <ol><li> 3 &#39;Ligasyon <ol><li> (Ağ / hac) PEG 8000, 0.5 ve 1.0 ul% 50: sıraya uygun olarak buz üzerinde, aşağıdaki bileşenleri monte956. l 10x RNA ligaz tamponu (500mM Tris-HCI pH 7.5, 100 mM MgCl2, 10 mM DTT), 1.0 ul, 3 &#39;Linker (100 uM), 0.5 ul RNaz inhibitörü, 0,5-8,0 ug toplam RNA, adenylated 0.5 ul T4 RNA ligaz, 2, 5 ul&#39;lik nihai bir hacme kadar nükleaz içermeyen H2O. </li><li> Çözelti girdaba çok viskoz olduğu gibi bağlı bir kez iyice karıştırılır PEG 8000 yüksek konsantrasyonu pipetleme tepkime karışımı ısıtılmış kapak ile, bir termal döngü reaksiyonu yerleştirin. 16 ° C&#39;ye kadar bloğu ayarlama, ve 4 saat süreyle inkübe edin. Not: Reaksiyon bağlanma verimliliği herhangi bir kayıp olmadan 20 ul bir son hacme kadar ölçeklendirilebilir. Çevre sıcaklığında 4 saat boyunca reaksiyona girmesi ya da gece boyunca 4 ° C &#39;de esas itibarıyla benzer bağlanma verimliliği elde edilir. </li></ol></li><li> Jel saflaştırma <ol><li> 3 &#39;Ligasyondan sonra, 2x RNA yükleyici tampon maddesinin eşit hacmi ile reaksiyona karıştırın. 5 dakika boyunca 70 ° C&#39;ye kadar ısı ve buz üzerinde serin ek. </li&gt; <li> PAGE arıtılması için bir% 15 poliakrilamid jeli üzerinde örnek çalıştırın. <ol><li> Adım 1.5&#39;te daha önce tarif edilene benzer bir şekilde, 7 M üre ihtiva eden bir% 15 poliakrilamid jelinde hazırlayın. </li><li> En az 30 dakika boyunca jel genişliği 24 V / cm jel ön çalıştırın. Öncesi dönemde aynı geriliminde güç kaynağı, floş kuyuları, yük örnek ve çalışma jel kapatın. </li><li> Örnek yükleyin ve Bromophenol mavi jel çıkıldı sadece kadar PAGE çalıştırın. Arzu edilen ürün, iksilen siyanol yakın hareket edecektir. </li></ol></li></ol></li><li> Boyama, Görselleştirme ve Eksizyon <ol><li> 15 dakika boyunca 1x çalışan tampon (TBE), 1x SYBR-altın ve kaya temiz tepsiye jel aparat ve yer jel sökmeye. </li><li> Boyanması çanak jel çıkarın ve temiz bir plastik wrap ile kaplı UV transilluminator&#39;de kutusunda görselleştirmek. Ligasyon ürünü, uzunluğu yaklaşık 45 nükleotid olacaktır. </li><li> Temiz bir Razo ile ligasyon ürünü içeren bölgeyi tüketim0.5 ml mikro-santrifüj tüpüne r bıçak ve yer. </li></ol></li><li> Ürün İzolasyon ve Yağış <ol><li> Pierce, 30 G iğne ile 0.5 ml mikrosantrifüj tüp alt ve ikinci bir temiz, düşük tutma deldi tüp yeri, 1.5 ml mikrosantrifüj tüp. ~ 15,000 x g&#39;de 5 dakika boyunca santrifüje. </li><li> Görme tüm jel dilim iç tüp delikten hareket ettiğini teyit etmektedir. Gerekirse, yakın deliğe bazı jel parçaları itmek için temiz bir pipet kullanın. </li><li> Boş 0.5 mi tüp atılır ve akrilamid bulamaca yüksek tuzlu kolon tamponu 400 ul (25 mM Tris-HCI pH 7.5,% 0.1 SDS HSCB 400 mM NaCl) ilave edin. Gece boyunca 4 ° C &#39;de kaya tüpü (s). </li><li> Geniş çaplı bir pipet kullanarak 0.22 mikron selüloz asetat spin kolon bulamaç aktarın. ~ 15,000 x g&#39;de, oda sıcaklığında 3 dakika boyunca santrifüje uzaklıkta akrilamid jel matrisi tüm sıvı toplamak. </li><li> Temiz, düşük tutma sıvı transfer, 1.5 ml tüp içerir20 mg / ml glikojen 1 ul ing. Bir izopropanol eşit hacim ve 10/1 inci hacim sodyum asetat ekleyin. Tüpü birkaç kez baş aşağı çevirerek çözeltisi karıştırın ve 20 dakika boyunca ° C sıcaklıkta dondurucuya -80 hızlandırabilir. </li><li> 20 dakika boyunca 4 ° C &#39;de tam hızda santrifüje nükleik asidi: pelet. Süpernatantı ve% 70 etanol içinde yıkayın. Havada kurutma, oda sıcaklığında 10 dakika süre ile pellet halindedir ve 5 &#39;bağlama adımı doğrudan devam edin. </li></ol></li></ol> Bağlayıcı Hybrid 3. Bağlayıcı ligasyon 5 &#39;miRNA-3 Sonu&#39; <ol><li> Tanelenmiş nükleik asit için, aşağıdaki bileşenler eklemek: 2 ul PEG 8000 (% 50 ağırlık / hacim), 0.5 ul 10x RNA ligaz tamponu, 0.5 ul ATP (10 mM), 0.5 ul NN-RNA, oligo (100 uM), ve H 4 ul bir son hacim 2 O. NOT: 5&#39;-ters dideoksi-TCUACAGUCCGACGAUCNN3 &quot;ifadesi ve üretici tarafından HPLC ile saflaştırıldı &#39;NN-RNA, oligo&#39; in bileşimi olup. </li><li> Karıştırmakaşağı defalarca yukarı pipetleme ve bu örnek. Pelet tamamen yeniden solübilize olmuştur emin olmak için bir süre (5 dk) için yapın. Yeniden-solübilizasyon sorunlu ise, 37 ° C örnek kısaca (3-5 dk) ısı ve karıştırmak için pipetlenmesini devam. </li><li> RNaz inhibitörü ve T4 RNA ligaz 1 (5 birim): 1&#39;lik bir karışımı topak çözelti içinde geri sonra, bir 1 1 ul içeren temiz bir PCR tüpü aktarılmaktadır. Yine aşağı yukarı pipetleme karıştırın. </li><li> 4 saat 37 ° C&#39;de bir termal döngü reaksiyonu inkübe edin. CDNA sentezi hemen geçin. </li></ol> 4. ters transkripsiyon / cDNA Sentezi <ol><li> 5 Aşağıdaki bileşenleri ekleme ligasyon reaksiyonu: 2 ul 5X RT tampon maddesi, 0,75 ul 100 mM DTT, 1 ul Illumina RT primer 5&#39;-GCCTTGGCACCCGAGAATTCCA-3 &#39;(1 uM) ve 0.5 ul dNTP (10 mM) getirilmiştir. </li><li> 5 dakika boyunca 65 ° C&#39;ye kadar pipetleme ve ısı ile karıştırın. Sonra yavaş yavaş tezgah t üzerinde soğumasınıop. </li><li> Reaksiyon, buz üzerinde, oda sıcaklığında, bir yer tüpüne soğutuldu ve ters transkriptaz enzimi ve RNase inhibitör her biri 0.5 ul ekleyin sonra. Imalatçının talimatlarında belirtildiği uzatma aşaması için ısı döngüleyici içinde pipetleme ve yerine göre karıştırın. </li><li> Tamamlandığında eklenti nükleaz içermeyen H2O 5 ul 15 ul kadar cDNA kütüphanesinin son hacmi getirmek. </li></ol> 5. Kütüphane hazırlanması <ol><li> Endeksleme PCR için, buz üzerinde, aşağıdaki reaksiyon monte: 6 ul 5x Phusion Tampon, 1 ul dNTP (10 mM), 0.9 ul DMSO, 0.75 ul 5 &#39;primer (25 uM), 0.75 ul 3&#39; primer (25 uM), 3 ul cDNA kütüphanesi (girdi miktarına bağlı olarak değişken konsantrasyon), 0.5 ul Phusion Polimeraz (1 ünite), 30 ul&#39;lik nihai bir hacme kadar nükleaz içermeyen H2O. Şablonun ve sıralama platformu ile uyumluluğu sağlamak için primer dizileri seçinseçim. <ol><li> 15 saniye için 72 ° C &#39;de uzanmaktadır, birinci 4 çevrim için, 3 dakika boyunca 98 ° C de ilk denaturasyon 15 sn için 50 ° C&#39;de 15 saniye, tavlama için 98 ° C denatüre: PCR, aşağıdaki ayarlarla bir reaksiyondan . NOT: giriş malzemenin miktarına bağlı olarak, toplam PCR çevrim numaraları değişir. Tipik olarak, toplam RNA 1-2 ug bir giriş için, 13 toplam PCR döngüleri mıR-Dizi kütüphane oluşturmak için yeterlidir. </li><li> Tavlama ve uzatma çevrimi bir araya getirilmiş ve 30 sn için ve denatürasyon için 70 ° C sıcaklıkta gerçekleştirilmektedir, iki aşamalı bir şekilde, örneğin daha sonraki aşamalarda, durulamalar, 5-12, yürütmek tekrar 15 sn için 98 ° C &#39;dir. Not: Reaksiyon hacmi PCR programı duraklatma izin veren ve arzu edilen ürün bir görünüm için izlemek için önceden belirlenmiş bir döngü sayılarında, 10 ul bir kısım kaldırmak üzere 30 ul. Tipik haliyle, çevrimleri 10, 13 ve 16 toplam RNA numunesinin test için seçilir. </li></ol></li><li> Hazırlamak% 40 akrilamid ve 2 ml 10x TBE 1 ml, ve H2O 10 ml karıştırılarak% 8 denatüre edici olmayan akrilamid jeli. Daha sonra% 10 APS 100 ul ve TEMED 10 ul ekle. Jel dökün ve set edelim. <ol><li> 30 dakika boyunca jel genişliği, 12 V / cm jel ön çalıştırın. Floş kuyular. 10x yükleme tamponu 1 ul ekle (15% Ficoll 400, 66 mM EDTA, 20 mM Tris-HCI, pH 8.0,% 0.1 (ağ / mavi / hacim) bromofenol hac) SDS,% 0.01 () 10 ul ekle boyutları her çukuruna 5 mm x 1 mm x 15 mm numune. Tipik olarak, her iyi örneklerin 30 ul yükleyebilirsiniz. Bromofenol mavi boya sadece jel alt bitene kadar 1x TBE tamponu içinde, jel genişliği 10 V / cm &#39;de örnek jel üzerinde çalıştırıldı. </li><li> Leke ve aşama 2.3&#39;de tarif edildiği gibi jel görselleştirmek. </li><li> 146 baz çift bant pulu ve Bölüm 2.4 de tarif edilene benzer bir şekilde, HSCB 0.25 ml, gece boyunca tasfiye edilir. Bir izopropanol eşit hacimde ve 10/1 inci hacim sodyum asetat DNA hızlandırabilir. % 70 e yıkama peletTE tamponunda -metanol ve tekrar süspansiyon. </li><li> Üreticinin tavsiyelerine göre PicoGreen kullanarak kütüphane niceliğini. </li></ol></li></ol>

<ol>
	<li>Hafner, 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=RNA-ligase-dependent+biases+in+miRNA+representation+in+deep-sequenced+small+RNA+cDNA+libraries.">RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries.</a> RNA. 17 (9), 1697-1712 (2011).</li><li>Zhang, Z., Lee, J. E., Riemondy, K., Anderson, E. M., Yi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+RNA+cloning+enables+accurate+quantification+of+miRNA+expression+by+deep+sequencing.">High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing.</a> Genome Biology. 14 (10), 109 (2013).</li><li>Consortium, T. E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ENCODE+(ENCyclopedia+Of+DNA+Elements)+Project.">The ENCODE (ENCyclopedia Of DNA Elements) Project.</a> Science. 306 (5696), 636-640 (2004).</li><li>Consortium, T. E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2012).</li><li>Linsen, S. E. V., 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=Limitations+and+possibilities+of+small+RNA+digital+gene+expression+profiling.">Limitations and possibilities of small RNA digital gene expression profiling.</a> Nature Methods. 6 (7), 474-476 (2009).</li><li>Jayaprakash, A. D., Jabado, O., Brown, B. D., Sachidanandam, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+remediation+of+biases+in+the+activity+of+RNA+ligases+in+small-RNA+deep+sequencing.">Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing.</a> Nucleic Acids Research. 39 (21), 141 (2011).</li><li>McLaughlin, L. W., Romaniuk, E., Romaniuk, P. J., Neilson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Acceptor+Oligoribonucleotide+Sequence+on+the+T4+RNA+Ligase+Reaction.">The Effect of Acceptor Oligoribonucleotide Sequence on the T4 RNA Ligase Reaction.</a> European Journal of Biochemistry. 125 (3), 639-643 (1982).</li><li>Zhuang, F., Fuchs, R. T., Sun, Z., Zheng, Y., Robb, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+bias+in+T4+RNA+ligase-mediated+3&#8242;-adapter+ligation.">Structural bias in T4 RNA ligase-mediated 3&#8242;-adapter ligation.</a> Nucleic Acids Research. 40 (7), 54 (2012).</li><li>Harrison, B., Zimmerman, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer-stimulated+ligation:+enhanced+ligation+of+oligo-+and+polynucleotides+by+T4+RNA+ligase+in+polymer+solutions.">Polymer-stimulated ligation: enhanced ligation of oligo- and polynucleotides by T4 RNA ligase in polymer solutions.</a> Nucleic Acids Research. 12 (21), 8235-8251 (1984).</li><li>Torchia, C., Takagi, Y., Ho, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Archaeal+RNA+ligase+is+a+homodimeric+protein+that+catalyzes+intramolecular+ligation+of+single-stranded+RNA+and+DNA.">Archaeal RNA ligase is a homodimeric protein that catalyzes intramolecular ligation of single-stranded RNA and DNA.</a> Nucleic Acids Research. 36 (19), 6218-6227 (2008).</li><li>Lau, N. C., Lim, L. P., Weinstein, E. G., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Abundant+Class+of+Tiny+RNAs+with+Probable+Regulatory+Roles+in+Caenorhabditis+elegans.">An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans.</a> Science. 294 (5543), 858-862 (2001).</li><li>Yi, 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=DGCR8-dependent+microRNA+biogenesis+is+essential+for+skin+development.">DGCR8-dependent microRNA biogenesis is essential for skin development.</a> Proceedings of the National Academy of Sciences. 106 (2), 498-502 (2009).</li><li>Leung, A. K. 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=Genome-wide+identification+of+Ago2+binding+sites+from+mouse+embryonic+stem+cells+with+and+without+mature+microRNAs.">Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs.</a> Nature Structural &amp; Molecular Biology. 18 (2), 237-244 (2011).</li></ol>

The authors declare no competing financial interests.

Bu tarifnamede anlatılan yöntemler, bağlama verimliliği, yani PEG, randomize bağlayıcıların kullanımı yüksek konsantrasyonlarda ve bağlayıcıların 2,6,9 yüksek konsantrasyonu en üst düzeye çıkarmak için birkaç anahtar değişkenlerden kullanır. Bu yaklaşım, total RNA örnekleri 2 güvenilir kantitatif sıralama kütüphaneleri izin verir. Biz giriş RNA&#39;nın çoklu titrasyonlarını yaptık ve önceki metodoloji (veriler gösterilmemiştir) 1-8 mikrogram aralığında to...

Yüksek verim sıralama tabanlı metodolojileri yaygın ölçüde biyolojik sistemlerin 3,4 moleküler karmaşıklığı anlayışımızı genişleyen son yıllarda birçok biyolojik numunelere uygulanmıştır. Ancak, yüksek sekanslama için RNA örneklerinin hazırlanması çoğu zaman, bu güçlü teknikleri potansiyel kullanımını sınırlayan çalışan yönteme özgü belirli bir yanlılığı kazandırır. Bu yöntem, belirli önyargıları iyi ligasyon-temelli, küçük RNA kütüphane hazırlıkları 1,2,5,6 için belgelenmiştir. Bu önyargıları 1,000 kat varyasyon çılgınca değişken ve hata eğilimli sıralama verilerinden miRNA bereket çıkarsama yapma, eşit molar sentetik miRNA&#39;ların için sayıları okur sonuçlanabilir. Faj türevli, T4 RNA ligazların özelliklerine odaklanarak çalışmalar enzimler yüksek verimli sekanslama deneylerde belirlendiği gibi yanlı kitaplıkları ortaya nükleotid bazlı tercihleri ​​7, olduğu belgelenmiştir adres <s1,2,8&gt; kadar. RNA, ligazlar tarafından kazandırılan hatalarını en aza indirmek için, birden fazla stratejiler kullanılmıştır; makromoleküler, 9 boğmakta ligasyon siteye 6 proksimalinde adaptör üzerinde nükleotid dizisi randomizing ve ligasyon adaptörü 2 yüksek konsantrasyonlarda istihdam. Bu üç yaklaşımların bir kombinasyonu sayesinde yüksek verimlilik sıralaması (Şekil 1) uyumlu küçük RNA kütüphanelerin tarafsız hazırlanması için bir iş akışı geliştirdik. Mevcut protokoller ve optimize edilmiş bir yöntemle arasında doğrudan karşılaştırmalar için, bizim son raporunda 2 başvurun. Bu optimize edilmiş yöntem her iki 3 &#39;ve 5&#39; aşamalarında% 95&#39;ten ligasyonu verim alınmasını sağlamaktadır, sentetik ve biyolojik örnekler 2 küçük RNA moleküllerinin tarafsız bağlanmasını izin verir.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name of Reagent/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:280px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">3&#39; Linker (5&#39; phosphorylated, 3&#39; blocked)</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">5&#39; Linker</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>5&#39; blocked, HPLC Purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">T4RNL2 (1-249 K227Q)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0351S</td>
			<td>Specialized for ligation of pre-adenylated DNA adapters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">10X Ligation Buffer (without ATP)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0351S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">10X Ligation Buffer (with ATP)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">RNaseOUT&#160;</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">10777-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Polyethylene Glycol (mol. Wt. 8000)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Nuclease-free water</td>
			<td style="width:175px;">Ambion</td>
			<td style="width:157px;">AM9937</td>
			<td>We have found water collected from a distillation apparatus to be of equvalent quality.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">T4RNL1</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Superscript III RT kit</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">18080-051&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Phusion PCR kit</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0530S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina RP1 Primer</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina RT Primer</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina Index Primer(s)</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">40% Acrylamide</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:157px;">BP14081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Urea</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">U6504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Ammonium persulfate</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tetramethyethylenediamine (TEMED)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">2X Denaturing RNA loading buffer</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0351S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Razor blades</td>
			<td style="width:175px;">VWR</td>
			<td style="width:157px;">55411-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">SpinX Centricon Tubes</td>
			<td style="width:175px;">Costar</td>
			<td style="width:157px;">CLS8161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Low Retention Microfuge tubes</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:157px;">02-681-320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sybr Gold</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">S-11494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Adenylation Kit</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">E2610L</td>
			<td></td>
		</tr>
	</tbody>
</table>

highly efficient ligation of small rna molecules for microrna quantitation by high-throughput sequencing

MiRNA klonlama ve yüksek verim sıralama, miR-Dizi, tek nükleotid çözünürlükte miRNA&#39;lar ölçmek için bir transcriptome geniş bir yaklaşım olarak tek başına duruyor adlandırılır. Bu teknik MiRNA moleküllere 3 &#39;ve 5&#39; oligonükleotid adaptörler takılarak miRNA&#39;lar yakalar de novo miRNA keşif sağlar. Güçlü yeni nesil dizileme platformları ile birleştirilmesi sonucu, miR-Seq miRNA biyoloji çalışmaya vesile olmuştur. Ancak, oligonükleotid ligasyon adımlarla tarafından tanıtıldı önemli önyargılar doğru bir kantitasyon aracı olarak istihdam edilmesini miR-seg engellemiştir. Önceki çalışmalar, mevcut miR-Dizi yöntemleri önyargıları genellikle bazı miRNA&#39;ların 1,2 1.000 kata kadar hataları ile yanlış miRNA miktar yol açtığını göstermektedir. RNA ligasyonu ile kazandırılan bu önyargıları gidermek için, biz de 3 &#39;ve 5&#39; ligasyon adımlar için% 95&#39;in üzerinde ligasyonu verimliliği ile sonuçlanan bir küçük RNA ligasyonu yöntemi geliştirdik. Bu im Kıyaslamasürekli beklenen değerden az iki kat sapmayla numaraları okur verir, eşit molar veya farklı olarak karışık sentetik miRNA&#39;lar kullanarak kitaplık inşaat yöntemi olduğunu kanıtladı. Ayrıca, bu yüksek verimli miR-Dizi yöntem in vivo toplam RNA örneklerinde 2 doğru genom miRNA profilleme izin verir.

Yukarıdaki yöntemi için beklenen sonuçlar, ilk (Şekil 2) Mde RNA ligaz ile PCP tabi tutuldu DNA oligonükleotidin büyüklüğünde tek bir nükleotid vardiya (artış) gözlenmesi gerekir. 3 &#39;bağlanmasından sonra, akrilamid jel görselleştirme (bakınız Şekil 3), jel, 100-300 nükleotid bölgesinde barizdir net, yüksek molekül ağırlıklı bant gösterir. Bu kullanılan toplam RNA numunesi yüksek kalitede olduğunu gösterir (bozulmuş değil). İkinci olarak...

Watch this Scientific Journal Video about Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing at JoVE.com

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

Yüksek Verim Dizilimine tarafından MicroRNA ölçümü için Küçük RNA molekülleri, yüksek Verimli ligasyonu

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

highly-efficient-ligation-small-rna-molecules-for-microrna

Research

JoVE Journal

Biology

11.8K Görüntüleme.  University of Colorado, Boulder. MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs. 

Video: Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

Gramicidin tabanlı Floresan Testi; çift katlı lipid Özellikleri değiştirmek için Küçük Moleküller Potansiyel Belirleme

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

Biyoloji

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

İçte düzeltici Potasyum Kanallarının Küçük moleküllü Modülatörler için Yüksek throughput Eleme

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / Cas Sistemleri Endonükleazlar için Substrat Üretimi

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Yüksek verim SHAPE kullanarak RNA İkincil Yapısı Tahmin

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Yüksek verim Sıralama için Yüksek Kalite Kromatin Immunoprecipitation DNA Şablon üretimi (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebra balığı Embriyolar kullanılarak Yüksek verim Yaklaşıyor A Manuel Küçük Molekül Ekran

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Post-transkripsiyonel ayarlı genlerin kimyasal Modülatörler için yüksek verimli tarama

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Otomatik Modüler Yüksek Verimli Ekzopolisakkarit Tarama Platformu Oldukça hassas Karbonhidrat Parmak İzi Analizi ile birleştiğinde

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID seq: Adenin-metillenmiş DNA Fragments Yüksek Verimli Dizileme tarafından Protein-DNA Etkileşimleri genom Haritalama

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

Yüksek verimlilik sıralaması kullanılması Tüm-Protein Doygunluk Mutagenez Kütüphaneleri Fonksiyonel Değerlendirme Protokolü

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...