Biz Kimyasal Bilimler, Yer Bilimleri ve Biyolojik Bilimler Bölümü, Temel Enerji Bilimler, Fen Dairesi, ABD Enerji Bakanlığı (ödül sayısı DE-FG02-91ER20021) ve Ulusal Bilim Vakfı (MCB 0.948.584) (FB) Müdürlüğü tarafından destek için minnettarım. Biz yazının düzenleme için Bayan Karen Kuş minnet borçluyuz.

1. EMS Tedavi Arabidopsis thaliana tohumu T / A mutasyonlar 5-7 C / G elde genomunun C-etmek-T içinde değişikliklerin indüklemektedir mutajen madde etil metan sülfonat (EMS) 3,4 olarak kullanılarak mutagenized edilir. <ol><li> Organel floresan işaretleyici (özellikle, bu çalışmada ssGFPHDEL (sinyal sekansı-GFP-HDEL tetrapeptide) bir ER belirteç olarak kullanılmıştır) taşıyan 0.8 g Arabidopsis tohumları (~ 40.000 tohum) tartılır. </li><li> 50 ml falcon tüp tohumları aktarın, sonra 25 ml saf su ekleyin. </li><li> % 0.2 (v / v) etil metan sülfonat ekleyin. </li><li> Düşük hızda karıştırıcı nutating 16 saat boyunca inkübe. </li><li> Sıvı aspire ve 1.0 M NaOH EMS inaktive içeren bir şişeye içine atın. </li><li> Yakın, tohum içeren Falcon tüpüne 25 ml su ekleyin ve 5 kez ters tohum yıkamak, tüm tohumları çökene kadar bekleyin, sonra 1.0 M Na içine su aspire ve atın<html> Şişesi OH. </li><li> 10 kata kadar yıkama işlemi tekrarlayın. </li><li> Nihai yıkamadan sonra, su minimal miktarda tohumların yeniden askıya. </li><li> % 10 çamaşır suyu 25 ml ekleyerek tohum sterilizasyon geçin, 30 saniye boyunca kuvvetlice çalkalanır. Daha sonra çamaşır suyu dökünüz ve steril su 25 ml ile yıkayın, tohumlar dibe edelim. Steril suyu dökün ve 25 ml% 70 etanol ekleyin. 30 s için tüpleri çalkalayın. Tohumlar dibe edelim. Etanol kapalı dökün ve steril su 25 ml ile yıkayın. Sonra 3 MM filtre kağıdı içeren plastik Petri tabağına tohumları dökün ve kaputun altında kurumaya bırakın, iki kez steril su ile yıkama tekrarlayın. 2 gün süre ile 4 ° C&#39;de muhafaza edilmelidir. </li><li> ½ MS phytagel (Murashige ve Skoog orta yarısı konsantrasyonu), 150 mm petri (plaka için ~ 250-300 tohumları) üzerine Plaka tohumları. </li><li> 2 hafta için plaka üzerinde M1 tohumlar büyüyecek, daha sonra toprağa nakli. </li><li> M2 hatları (1000 bağımsız hat) oluşturmak için bireysel M1 bitkilerden M2 tohumlar toplayın. </li></ol>M2 ve M3 Populasyonlarının ove_title &quot;&gt; 2. Konfokal Tarama Bu bölümde daha önce 8 açıklandığı gibi bir konfokal veya floresan mikroskobu ile fide gözlem açıklar. <ol><li> Her M2 hattan altmış tohumları plastik Petri, 7 ila 10 gün, ½ MS phytagel için yetiştirilen; aynı plaka üzerinde de (kontrol), EMS-tedavi edilmemiş 5 fideleri yetiştirilmektedir. </li><li> Beş on kotiledon bir mikroskop lamı üzerine objektif (40X) doğru eksen dışı tarafı ile monte edilmiş ve bir coverslip ile birlikte verilmektedir. </li><li> Her kotiledon organelidir işaretleyici her değişmiş Subselüler dağıtılması için floresans altında, kortikal gelen medyal bölgeye, görülmektedir. </li><li> Pozitif bitkiler toprak nakil vardır ve bunların tohumları toplanır ve M3 nesil mutant fenotip onaylamak için tekrar taranmaktadır. </li><li> Muhtemelen birlikte bir yabani tip genomuna backcrossing yoluyla mutasyonlar arka kirletici en az üç kez çıkarınİstediğiniz organel floresan işaretleyici ntaining. <ol><li> Anne bitki, ince makas veya forseps kullanarak olgun siliques kaldırmak ve açık çiçekler. </li><li> Meristemlerinde çok küçük tomurcuklar çıkarın. </li><li> Yaprakları ve bir çiçek tomurcuğu açın ve tüm anter kaldırmak için çanak yaprakları arasında forseps bir çift ucu yerleştirin. </li><li> Forseps bir çift kullanarak baba bitki açık bir olgun çiçek alıp hadım bitkinin tepeciklerdeki anter ovalayın. </li></ol></li></ol> 3. Haritalama Bu bölümde, geleneksel haritalama yöntemleri 10,11 ile kıyaslandığında daha hızlı Borevitz 9 değiştirilmiş bir protokolü kullanan bir resesif mutasyon eşlemek aslında açıklamaktadır. Bu yaklaşım tek bir tahlil 12 sayısız tek özelliği polimorfizmleri (SFP) tespit etme yeteneğine sahip yüksek yoğunluklu oligonükleotid diziler kullanın. Affymetrix Arabidopsis ATH1 GeneChip dizi kullanmak mümkünyaklaşık 24.000 gen analiz. Mutasyon gösteren F 2 bireylerin bir havuz aynı ayırma nüfus içinde toplanan yabani tip F 2 bitkilerin bir havuz ile karşılaştırılır. Daha sonra, mutasyon mutant havuzu mutant genotip allelleri için zenginleştirilmiş bölgede eşlenecek ve dolayısıyla aynı bölgede yabani tip havuzu vahşi tip üst allelleri 13 zenginleştirilmiş sonuçlanacaktır. <ol><li> Genomik DNA (3 mg) bir Qiagen DNeasy Bitki Mini Kit kullanılarak homozigot mutant Columbia (M3) elde edilir ve Illumina Genom Analyzer II (GA II) 14 sıralaması için sunulmuştur. </li><li> Homozigot mutant Columbia (M3) bir eşleme nüfus üretmek için Landsberg erecta ile çarpı işareti vardır. </li><li> Haritalama anormal fenotip ve vahşi tip fenotipi ile F2 bitkilerin aynı sayıda gösteren 70 kadar 100 F2 bireyler üzerinde yapılır. </li><li> Bir delik açma kullanarak yaprak disk her bitki (0,60 mm) toplayın.yaprak disk DNA benzer miktarda olduğundan emin olmak için aynı yaştaki yapraklarından elde toplanmalıdır. Örnekler genomik ekstraksiyon ayrı ayrı veya gruplar için işlenebilir. Bu durumda, bu sonuçta bir genomik DNA elde edilmesi için olan hangi az Eppendorf tüpler sahip olacak şekilde her bir Eppendorf tüpüne için 5-10 örnekleri toplamak mümkündür. </li><li> MasterPure Bitki Yaprak DNA izolasyon kiti (Epicentre) genomik DNA elde etmek için kullanılmaktadır. Her bir numune elde edilen bir genomik DNA nanodrop ile ölçülür. </li><li> Her örnek genomik DNA aynı miktar daha sonra bitki DNA&#39;ların 300 ng (~ 30 uL) toplam kadar birlikte alınır; 60 uL 2.5X rastgele primerler çözümü eklemek [125 mM Tris-HCl (pH 6.8), 12.5 mM MgCl2, 25 mM 2-merkaptoetanol, 750 ug / ml oligodeoksiribonükleotid primerler (rastgele octamers)] (Bioprime kiti) ve 42 uL su (nihai hacim 132 uL). </li><li> Denatüre&gt; 95 ° C de DNA&#39;lar 5-10 dakika için. </li><li> Buz üzerinde serin. </li><li> Her denatu içinkırmızı bir DNA Biotin dCTP [1 mM biyotin-14-dCTP, 1 mM dCTP, 2 mM dATP, dGTP 2 mM, 10 mM Tris-HCl içinde 2 mM dTTP (pH 7.5), 1 mM Na2EDTA] ile 15 uL 10X dNTP karıştırılır (Bioprime kiti) ve 3 uL Klenow polimeraz (Bioprime kiti) ile tamamlamak. </li><li> 25 ° C&#39;de bir gece boyunca inkübe </li><li> Karması; Ertesi gün, 15 uL 3 M NaOAc ve 400 uL soğuk% 100 EtOH ekleyin. </li><li> 1 saat -80 ° C&#39;de inkübe edin, sonra 15 dakika 20,500 xg&#39;de spin süpernatant kaldırmak ve 500 uL soğuk% 75 EtOH ile yıkayın. </li><li> 10 dakika süreyle 20,500 x g&#39;de dönerler. </li><li> 100 uL su içinde 10 dakika ve Pastör pipetiyle için 37 ° C&#39;da kuru bir DNA peletleri. </li><li> Bir jel (Şekil 2) verim ve kalite kontrol etmek için 5 uL kullanın. </li><li> Için yabani tip ve mutant GeneChip Arabidopsis ATH1 Genom Array Hibridizasyon için reaksiyon etiketleme 95 uL gönder. </li><li> R yazılım (kullanarak diziler taranır ve elde. CEL dosyaları analiz edilir <a href="http://www.r-project.org/">http://www.r-project.o</a>rg /). </li><li> R yazılımı yükleyin, sonra programı açın ve aşağıdaki dize yapıştırın: kaynağı (&quot; <a href="http://bioconductor.org/biocLite.R">http://bioconductor.org/biocLite.R</a> &quot;) biocLite () Sonra geri dönüş tuşu-Bu standart Bioconductor paketleri (kuracaktır basın <a href="http://www.bioconductor.org">http://www.bioconductor.org</a> ). </li><li> Masaüstünde yeni bir klasör, aşağıdaki web sitesinden (indirdiğiniz <a href="http://www.naturalvariation.org/methods">http://www.naturalvariation.org/methods</a> ) dosyaları: readcel.R SFP.R Map.R ath1V5.RData ColLerCEL.zip Dosya ColLerCEL.zip ambalajsız ve sonuç dosyaları yeni klasöre yapıştırılır. </li><li> Wildtype.CEL ve mutant.CEL içine GeneChip deneyden elde edilen veriler yeniden adlandırın. </li><li> Lütfen GeneChip veriler artık kopyalayın (wildtype.CEL veKlasör içine mutant.CEL). </li><li> Açık R, Mac için): ardından &quot;Çeşitli&quot; ve &quot;Değişim çalışma dizini&quot; seçeneğine tıklayın. b) PC için: tıklayın &quot;Dosya&quot; ardından &quot;Değişim dizini&quot; </li><li> Mac için: a) dosyalarını içeren klasörü seçin. &quot;Çalışma Alanı&quot;, &quot;Yük çalışma alanı dosyasını tıklatın:&quot; sonra &quot;Dosya, Yük çalışma alanı&quot; &quot;PC için basıp ath1V5.RData, b) Yukarıda dosyalarını içeren klasörü seçin,&quot; ve ath1V5.RData seçin. </li><li> Açık readCEL.R Not Defteri&#39;ni kullanarak ve R. için tüm metni kopyalamak </li><li> Açık SFP.R Not Defteri&#39;ni kullanarak ve R. için tüm metni kopyalamak </li><li> Açık Map.R Not Defteri&#39;ni kullanarak ve R. için tüm metni kopyalamak </li><li> Konsol penceresinde aşağıdaki iletiyi görürsünüz: Kromozom x Mb yz sınırlar TAIR arayın genleri bu linki yapıştırın 3 <a href="http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y&amp;end=z">http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y</a>Ve sonunda = z </li><li> İnternet tarayıcınıza linki kopyalayıp yapıştırın. Bir pencere açılacak ve dosyayı açmak veya kaydetmek için sorar; kaydetmek seçmelisiniz. Bir dosya adı seçin ve uzantısı ekleyiniz &quot;. Xls&quot; Kaydet öğesini tıklatın. </li><li> Bu dosyayı açmaya Eğer mutant için koordinat (sonucu grafiksel Şekil 3&#39;te temsil edilir) bulabilir &quot;. Xls&quot;. </li><li> Monte Illumina olduğu eşleşen alanı içinde mutant genomunun okur tek nükleotid polimorfizmleri arasında belli EMS geçişler 4 tanımlamak. Standart prosedürler kullanılarak 15 vahşi tip gen (ler) ile dönüşümün mutant fenotip tamamlar. </li></ol> 4. Temsilcisi Sonuçlar Şekil 1 konfokal mikroskopi kullanılarak tarama sekretuar yolunun bir mutant belirlenmesi için kullanılan bir yaklaşım göstermektedir. Şekil 2 dizi hibridizasyon için etiketli genomik DNA tipik bir hazırlık gösterir. Şekil 3, GeneChip Arabidopsis ATH1 Genom Array elde edilen veriler sunulmuştur analiz ettikten sonra beklenen tipik bir sonucu olarak. <img alt="Şekil 1" src="/files/ftp_upload/3809/3809fig1.jpg" /> SsGFPHDEL (ER işaretleyici) (1) ifade Şekil 1. Arabidopsis transgenik EMS muamele tesislerinde (2) için yeterli tohum üretmek için yetiştirildi. EMS ile muamele edilen tohumlarda sonra M1 bitkilerinin (3) oluşturmak için ekilmiştir. Her M1 bitki farklı bir çizgi temsil eder ve tohum her birinden ayrı ayrı toplandı. M2 tohum ½ MS substrat (4) üzerine kaplama ve sonra konfokal mikroskopi (5) ile ER morfolojisi kusurlar için tarandı. Tarama sırasında biz yabani tip ER morfolojisi (6) ve kusurlu ER fenotipleri (7) göstermek bitkiler ve koruyan bitkiler bulundu. Bu bitkiler M3 nesli elde etmek ve fenotipi (8) onaylamak için yetiştirilmiştir. M3 bitki genomik DNA sekanslama Solexa Illumina (a) için kullanıldı. Aynı bitkiAyrıca, F2 eşleme nüfusu (b) elde etmek için Ler-WT ile melezleme için kullanılmıştır. <img alt="Şekil 2" src="/files/ftp_upload/3809/3809fig2.jpg" /> Şekil 2,. Bioprime rastgele etiketleme reaksiyonları (100 uL 5 ul) 1 agaroz jel% üzerine yüklendi. Lane vahşi tip F2 bitkilerin bir havuzdan ve şerit b mutant F2 bitkilerin bir havuz değil. (Marker NEB gelen, 1 Kb DNA merdiveni-N3232) olduğunu. <img alt="Şekil 3" src="/files/ftp_upload/3809/3809fig3.jpg" /> Şekil 3. Şekil GeneChip Arabidopsis ATH1 Genom Array Hibridizasyon kullanarak Col-0 mutasyon eşleme örneği temsil eder. Bu örnekte mutasyonu dikey çubuklarla kromozom 1, sınırlandırılmış bulunmaktadır. Yatay çubuklar tespiti için eşiklerin temsil eder.

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Biz ifşa hiçbir şey yok.

Burada endomembrane mutantların tanımlanması için bir konfokal mikroskopi tabanlı tarama nitelendirdi. Bu yaklaşım kolaylıkla spesifik floresan protein belirteçler mevcut olduğu hücrenin diğer organelleri kadar uzatılabilir. Ekran hedef organel veya işaretleyici içeren gerekiyordu değil organeller için ya floresan işaretleyici bir anormal dağılım gösteren mutantların tanımlanması dayanmaktadır. Sırasıyla, bu mutantlar işaretleyici subcompartimentalize için organel yeteneği ya tehlikeye dü...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Ethylmethane sülfonat </td><td> Sigma </td><td> M0880 </td></tr><tr><td> NaOH </td><td> JT Baker </td><td> 3722-05 </td></tr><tr><td> Murashige Skoog bazal orta w Gamborg vitaminler </td><td> Bitkisel teknolojileri Laboratuvar </td><td> M404 </td></tr><tr><td> Phytagel </td><td> Sigma </td><td> P8169-1Kg </td></tr><tr><td> RNeasy Mini kiti </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> Ana saf bitki yaprak DNA saflaştırma kiti </td><td> Epicentre </td><td> MPP92100 </td></tr><tr><td> Bioprime DNA etiketleme sistemi </td><td> Invitrogen </td><td> 18094-011 </td></tr><tr><td> Alkol 200 kanıtı </td><td> Dekan laboratuvarları inc. </td><td> 2716 </td></tr><tr><td> NaOAc </td><td> JT Baker </td><td></td></tr><tr><td> Gen çip Arabidopsis ATH1genom dizisi </td><td> Affymetrix </td><td> 900385 </td></tr><tr><td> Falcon tüpler 50 mL </td><td> corning </td><td> 430290 </td></tr><tr><td> Eppendorf Tüp 1,5 ml </td><td></td><td></td></tr><tr><td> 90mm Filtre kağıdı </td><td> Whatman </td><td> 1001090 </td></tr><tr><td> Analitik Terazi </td><td> Mettler Toledo AB54-S </td><td> na </td></tr><tr><td> Nutating (dalga) çalkalayıcı </td><td> Heidolph Polymax 1040 </td><td> na </td></tr><tr><td> Santrifüj </td><td> Eppendorf 5417-R </td><td> na </td></tr></tbody></table>

fluorescence-microscopy screening and next-generation sequencing: useful tools for the identification of genes involved in organelle integrity

Bu protokol Arabidopsis fidelerinin bir floresan mikroskop tabanlı tarama açıklanır ve sekretuar yolun belirli bir etiketli floresan marker Subselüler dağılımını değiştirebilir resesif mutasyonlar eşlemek anlatılmaktadır. Arabidopsis nedeniyle genom büyüklüğü genetik çalışmalar için güçlü bir biyolojik model, nesil zaman ve krallıkları arasında moleküler mekanizmaları korunması. Moleküler belirteçler dayalı geleneksel yönteme alternatif olarak mutasyon eşlemek için bir yaklaşım olarak genotipleme dizi nispeten hızlı olduğu için avantajlı olduğunu ve gerçekten çok kısa süre içerisinde birçok mutantların haritalama izin verebilir. Bu yöntem, bitkilerde herhangi bir organelidir bütünlüğünü etkileyebilir proteinlerin tanımlama sağlar. Burada bir örnek olarak, endoplazmik retikulum (ER) bütünlüğü için önemli gen haritası için bir ekran öneriyoruz. Bizim yaklaşımımız, ancak kolayca diğer bitki hücre organelleri uzatılabilir(Örneğin 1,2 bakınız), ve böylece diğer hücre içi yapıları düzenleyen moleküler temelini anlamaya yönelik önemli bir adımı temsil etmektedir.

Watch this Scientific Journal Video about Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity at JoVE.com

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

Hücre biyolojisinde temel bir arayış ökaryotik hücrelerin yapmak organellerin kimliği altında yatan mekanizmaları belirlemektir. Burada floresan mikroskobu ve yeni nesil dizileme araçlarını kullanarak bitki organel morfolojik ve fonksiyonel bütünlüğü sorumlu genleri tanımlamak için bir yöntem öneriyoruz.

Floresans-mikroskopi Tarama ve nesil Sekanslama: organel Bütünlüğü ile ilgilendim Genlerin belirlenmesi için Faydalı Araçlar

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

fluorescence-microscopy-screening-next-generation-sequencing-useful

Research

JoVE Journal

Biology

12.2K Görüntüleme.  Michigan State University. Hücre biyolojisinde temel bir arayış ökaryotik hücrelerin yapmak organellerin kimliği altında yatan mekanizmaları belirlemektir. Burada floresan mikroskobu ve yeni nesil dizileme araçlarını kullanarak bitki organel morfolojik ve fonksiyonel bütünlüğü sorumlu genleri tanımlamak için bir yöntem öneriyoruz.

Video: Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Zaman atlamalı Görüntüleme siRNA Transfeksiyon sonra Mitoz

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Biyoloji

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Nematod Vücut büyüklüğü Yönetmelik ilgilendim Genler için Ekran RNA-aracılı Girişim Besleme Strateji Kullanımı<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

16S Ribozomal RNA Gen amplikonlarının nesil Sıralama

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

DNA Metilasyon Analizi nesil Dizilimine tarafından hedef

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

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 ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviral Entegrasyon Siteleri Amplifikasyon, Yeni Nesil Sıralama ve Genomik DNA Haritalama

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Interactome-Seq: Domainome kitaplığı inşaat, doğrulama ve seçim fajının görüntü ve sonraki nesil sıralama için bir protokol

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

Kene Microbiome karakterizasyonu tarafından yeni nesil 16S rRNA Amplicon sıralama

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

İki adımlı PCR ve Yeni Nesil 16S rRNA Gen Sıralaması Kullanarak Mikrobiyota Analizi

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

Yeni Nesil Sıralama için Manyetik Boncuk Bazlı Sivrisinek DNA Ekstraksiyon Protokolü

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...