Name: identification of circular rnas using rna sequencing
Uploaded: 2019-11-14T11:00:00
Description: Watch this Scientific Journal Video about Identification of Circular RNAs using RNA Sequencing at JoVE.com

Biz Banner Güneş Sağlık Araştırma Enstitüsü Beyin ve Vücut Bağışı Programı (BBDP) Sun City, Arizona insan beyin dokularının sağlanması için müteşekkiriz. BBDP Ulusal Nörolojik Bozukluklar ve İnme Enstitüsü (U24 NS072026 Parkinson Hastalığı ve İlişkili Bozukluklar için Ulusal Beyin ve Doku Kaynağı), Ulusal Yaşlanma Enstitüsü (P30AG19610 Arizona Alzheimer Hastalığı Çekirdek Merkezi), Arizona Sağlık Hizmetleri Bölümü (sözleşme 211002, Arizona Alzheimer Araştırma Merkezi), Arizona Biyomedikal Araştırma Komisyonu (sözleşmeler 4001, 0011, 05-901 ve 1001 Arizona Parkinson Hastalığı Konsorsiyumu için) ve Michael J. Parkinson Araştırma Fox Vakfı27. Bu çalışma aynı zamanda DHS ve Arizona Eyaleti (ADHS hibe # ADHS14-052688) tarafından desteklenmiştir. Ayrıca Andrea Schmitt (Banner Araştırma) ve Cynthia Lechuga (TGen) idari destek için teşekkür ederiz.

Bu araştırma, insan refahı için tüm kurumsal, ulusal ve uluslararası kurallara uygun olarak gerçekleştirilmiştir. Beyin dokuları Sun City, AZ Banner Güneş Sağlık Araştırma Enstitüsü Beyin ve Vücut Bağışı Programı elde edildi. Beyin ve Vücut Bağışı Programı'nın faaliyetleri Western Kurumsal İnceleme Kurulu (WIRB protokolü #20120821) tarafından onaylanmıştır. Tüm konular veya yasal temsilcileri bilgilendirilmiş onayı imzaladı. Ticari (beyin dışı) biyonumuneler Proteogenex'ten sat...

<ol>
	<li>Salzman, J., Gawad, C., Wang, P. L., Lacayo, N., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNAs+are+the+predominant+transcript+isoform+from+hundreds+of+human+genes+in+diverse+cell+types.">Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types.</a> PloS One. 7 (2), e30733 (2012).</li><li>Jeck, W. 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=Circular+RNAs+are+abundant,+conserved,+and+associated+with+ALU+repeats.">Circular RNAs are abundant, conserved, and associated with ALU repeats.</a> RNA. 19 (2), 141-157 (2013).</li><li>Memczak, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNAs+are+a+large+class+of+animal+RNAs+with+regulatory+potency.">Circular RNAs are a large class of animal RNAs with regulatory potency.</a> Nature. 495 (7441), 333-338 (2013).</li><li>Sanger, H. L., Klotz, G., Riesner, D., Gross, H. J., Kleinschmidt, A. 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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type+specific+features+of+circular+RNA+expression.">Cell-type specific features of circular RNA expression.</a> PLoS Genetics. 9 (9), e1003777 (2013).</li><li>Du, W. W., 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=Foxo3+circular+RNA+promotes+cardiac+senescence+by+modulating+multiple+factors+associated+with+stress+and+senescence+responses.">Foxo3 circular RNA promotes cardiac senescence by modulating multiple factors associated with stress and senescence responses.</a> European Heart Journal. 38 (18), 1402-1412 (2016).</li><li>Capel, B., 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=Circular+transcripts+of+the+testis-determining+gene+Sry+in+adult+mouse+testis.">Circular transcripts of the testis-determining gene Sry in adult mouse testis.</a> Cell. 73 (5), 1019-1030 (1993).</li><li>Hansen, T. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+landscape+of+circular+RNA+expression+in+the+human+heart.">A landscape of circular RNA expression in the human heart.</a> Cardiovascular Research. 113 (3), 298-309 (2016).</li><li>Zhong, Z., Lv, M., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+differential+circular+RNA+expression+profiles+reveals+the+regulatory+role+of+circTCF25-miR-103a-3p/miR-107-CDK6+pathway+in+bladder+carcinoma.">Screening differential circular RNA expression profiles reveals the regulatory role of circTCF25-miR-103a-3p/miR-107-CDK6 pathway in bladder carcinoma.</a> Scientific Reports. 6, 30919 (2016).</li><li>Gao, Y., Wang, J., Zhao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CIRI:+an+efficient+and+unbiased+algorithm+for+de+novo+circular+RNA+identification.">CIRI: an efficient and unbiased algorithm for de novo circular RNA identification.</a> Genome Biology. 16, 4-014-0571-0573 (2015).</li><li>Wang, K., 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=MapSplice:+accurate+mapping+of+RNA-seq+reads+for+splice+junction+discovery.">MapSplice: accurate mapping of RNA-seq reads for splice junction discovery.</a> Nucleic Acids Research. 38 (18), e178 (2010).</li><li>Szabo, 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=Statistically+based+splicing+detection+reveals+neural+enrichment+and+tissue-specific+induction+of+circular+RNA+during+human+fetal+development.">Statistically based splicing detection reveals neural enrichment and tissue-specific induction of circular RNA during human fetal development.</a> Genome Biology. 16, 126-015-0690-0695 (2015).</li><li>Cheng, J., Metge, F., Dieterich, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+identification+and+quantification+of+circular+RNAs+from+sequencing+data.">Specific identification and quantification of circular RNAs from sequencing data.</a> Bioinformatics. 32 (7), 1094-1096 (2016).</li><li>Zhang, X. O., 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=Complementary+sequence-mediated+exon+circularization.">Complementary sequence-mediated exon circularization.</a> Cell. 159 (1), 134-147 (2014).</li><li>Rybak-Wolf, A., 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=Circular+RNAs+in+the+mammalian+brain+are+highly+abundant,+conserved,+and+dynamically+expressed.">Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically expressed.</a> Molecular Cell. 58 (5), 870-885 (2015).</li><li>Ji, P., 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=Expanded+Expression+Landscape+and+Prioritization+of+Circular+RNAs+in+Mammals.">Expanded Expression Landscape and Prioritization of Circular RNAs in Mammals.</a> Cell Reports. 26 (12), 3444-3460 (2019).</li><li>Hansen, T. B., Ven&#248;, M. T., Damgaard, C. K., Kjems, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+circular+RNA+prediction+tools.">Comparison of circular RNA prediction tools.</a> Nucleic Acids Research. 44 (6), e58 (2016).</li><li>Zeng, X., Lin, W., Guo, M., Zou, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+overview+and+evaluation+of+circular+RNA+detection+tools.">A comprehensive overview and evaluation of circular RNA detection tools.</a> PLoS Comput Biol. 13 (6), e1005420 (2017).</li><li>Sekar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACValidator:+a+novel+assembly-based+approach+for+in+silico+validation+of+circular+RNAs.">ACValidator: a novel assembly-based approach for in silico validation of circular RNAs.</a> bioRxiv. , (2019).</li><li>Westholm, J. O., 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+analysis+of+drosophila+circular+RNAs+reveals+their+structural+and+sequence+properties+and+age-dependent+neural+accumulation.">Genome-wide analysis of drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation.</a> Cell Reports. 9 (5), 1966-1980 (2014).</li><li>Szabo, L., Salzman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+circular+RNAs:+bioinformatic+and+experimental+challenges.">Detecting circular RNAs: bioinformatic and experimental challenges.</a> Nature Reviews Genetics. 17 (11), 679-692 (2016).</li><li>Zheng, Y., Ji, P., Chen, S., Hou, L., Zhao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+full-length+circular+RNAs+enables+isoform-level+quantification.">Reconstruction of full-length circular RNAs enables isoform-level quantification.</a> Genome Medicine. 11 (1), 2 (2019).</li><li>Beach, T. 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Bu çalışmada, circRNA sıralama kitaplıklarının inşası için circRNA zenginleştirme protokolünü optimize etmek amacıyla ticari olarak kullanılabilen iki kütüphane hazırlama kitleri, ön işlem seçenekleri ve giriş RNA miktarları test edilmiştir. Bu çalışmanın değerlendirmelerine dayanarak, circRNA sıralama kitaplıklarının oluşturulmasında önemli ve kritik adımlar ortaya çıkmıştır. Değerlendirmemiz, tespit edilen circRNA sayısının artmasıyla yansıtTığı üzere RNase R ön arı...

Dairesel RNA'lar (CircRNA'lar) ökaryotik transkripsiyon1,2,3'teyaygın olarak ifade edilen endojen, kodlamayan RNA'lardır. Onlar exons birbirlerine geri-splice oluşur ve bu nedenle başlangıçta eserler4,5birleştirme olarak kabul edildi . Ancak, son çalışmalar, circRNA'ların hücre tipini, dokuyu ve gelişim evresine özgü ekspresyonu3,6 ve evrimsel olarak korunmuş2,3olduğunu göstermiştir. Ayrıca, protein-protein etkileşimleri arabuluculuk yer almaktadır7, mikro-RNA (miRNA) bağlayıcı3,8,9,10, ve ebeveyn gen transkripsiyon11düzenlenmesi .
Klasik RNA diziliminde (RNA-seq), circRNA'lar mRNA'lar için poli-A seçimi sonucunda kütüphane inşaatı sırasında tamamen kaybolabilir veya düşük bollukları göz önüne alındığında izole edilmesi zor olabilir. Ancak, son circRNA karakterizasyon çalışmaları amacıyla circRNA2,12,13için zenginleştirmek için RNase R kullanarak bir ön tedavi adımı dahil var. RNase R, dairesel RNA yapılarını geride bırakarak lineer RNA'ları sindiren bir exoribonuclease'dir. CircRNA zenginleştirme protokolleri, rnase R ön işlem adımı olan ve olmayan iki ticari olarak kullanılabilen tüm transkripsiyon kitaplığı yapı kitlerinden elde edilen verilerin üretilmesi ve karşılaştırılması ve değişen miktarlarda toplam RNA girişi kullanılarak optimize edilmiştir (1 ila 4 g). Optimize edilmiş protokol daha sonra beş farklı beyin bölgesinde (beyincik [BC], inferior parietal lob [IP], orta temporal girus [MG], oksipital korteks [OC] ve üstün frontal girus [SF]) ve diğer dört doku tipi (karaciğer [LV], akciğer [LU], lenf nodu [LN] ve pankreas [PA]) arasında circRNA bolluğunu değerlendirmek için kullanıldı. RNA-seq kütüphaneleri sıralı olarak eşleştirilmiş ve veriler altı farklı circRNA tahmin algoritmaları kullanılarak analiz edildi: find_circ3, CIRI14, Mapsplice15, KNIFE16, DCC17, ve CIRCexplorer18. Analizlerimize göre, RNase R ön işlem ve 4 μg toplam giriş RNA ile mahsur toplam RNA kütüphane hazırlama kiti kullanılarak en yüksek sayıda benzersiz circNA tespit edilmiştir. Optimize edilmiş protokol burada açıklanmıştır. Daha önce bildirildiği gibi19,20, circRNA en yüksek zenginleştirme diğer doku tipleri ile karşılaştırıldığında beyinde gözlendi.

<table>
	<tbody>
		<tr>
			<td>1000 &#181;L pipette tips</td>
			<td>Rainin</td>
			<td>GP-L1000F</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;L pipette tips</td>
			<td>Rainin</td>
			<td>SR L 10F</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L pipette tips</td>
			<td>Rainin</td>
			<td>SR L 200F</td>
			<td></td>
		</tr>
		<tr>
			<td>2200 TapeStation Accessories (foil covers)</td>
			<td>Agilent Technologies</td>
			<td>5067-5154</td>
			<td></td>
		</tr>
		<tr>
			<td>2200 TapeStation Accessories (tips)</td>
			<td>Agilent Technologies</td>
			<td>5067-5153</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Film for Microplates</td>
			<td>VWR</td>
			<td>60941-064</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure XP Beads 450 mL</td>
			<td>Beckman Coulter</td>
			<td>A63882</td>
			<td>PCR purification</td>
		</tr>
		<tr>
			<td>Eppendorf twin.tec 96-Well PCR Plates</td>
			<td>VWR</td>
			<td>951020401</td>
			<td></td>
		</tr>
		<tr>
			<td>High Sensitivity D1000 reagents</td>
			<td>Agilent Technologies</td>
			<td>5067-5585</td>
			<td></td>
		</tr>
		<tr>
			<td>High Sensitivity D1000 ScreenTape</td>
			<td>Agilent Technologies</td>
			<td>5067-5584</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 2500 Sequencing System</td>
			<td>Illumina</td>
			<td>SY-401-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 3000/4000 PE Cluster Kit</td>
			<td>Illumina</td>
			<td>PE-410-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 3000/4000 SBS Kit (150 cycles)</td>
			<td>Illumina</td>
			<td>FC-410-1002</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 4000 Sequencing System</td>
			<td>Illumina</td>
			<td>SY-401-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq PE PE Rapid Cluster Kit v2</td>
			<td>Illumina</td>
			<td>PE-402-4002</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq Rapid SBS Kit v2 (50 cycle)</td>
			<td>Illumina</td>
			<td>FC-402-4022</td>
			<td></td>
		</tr>
		<tr>
			<td>Kapa Total RNA Kit</td>
			<td>Roche</td>
			<td>KK8400</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular biology grade ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP28184</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Assay Tubes</td>
			<td>Supply Center by Thermo Fischer</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA High Sense Assay Kit</td>
			<td>Supply Center by Thermo Fischer</td>
			<td>Q32854</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA cleanup and concentrator - 5</td>
			<td>Zymo</td>
			<td>RCC-100</td>
			<td>Contains purification columns, collection tubes</td>
		</tr>
		<tr>
			<td>RNAClean XP beads</td>
			<td>Beckman Coulter Genomics</td>
			<td></td>
			<td>RNA Cleanup beads</td>
		</tr>
		<tr>
			<td>Rnase R</td>
			<td>Lucigen</td>
			<td>RNR07250</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase 10,000 units</td>
			<td>ThermoFisher (LifeTech)</td>
			<td>18064014</td>
			<td></td>
		</tr>
		<tr>
			<td>TapeStation 2200</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td>Nucleic Acid analyzer</td>
		</tr>
		<tr>
			<td>TElowE</td>
			<td>VWR</td>
			<td>10128-588</td>
			<td></td>
		</tr>
		<tr>
			<td>TruSeq Stranded Total RNA Library Prep Kit</td>
			<td>Illumina</td>
			<td>20020596</td>
			<td>Kit used in section 3</td>
		</tr>
		<tr>
			<td>Two-Compartment Divided Tray</td>
			<td>VWR</td>
			<td>3054-1004</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Water</td>
			<td>Supply Center by Thermo Fischer</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal control RNA</td>
			<td>Agilent</td>
			<td>740000</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of circular rnas using rna sequencing

Dairesel RNA'lar (circRNA'lar), mikro-RNA (miRNA) düzenlemesi, protein-protein etkileşimlerinin arabuluculuğu ve ebeveyn gen transkripsiyonunun düzenlenmesi gibi işlevlerde yer alan kodlamayan RNA'lar sınıfıdır. Klasik yeni nesil RNA sıralamasında (RNA-seq), circRNA'lar genellikle mRNA kitaplıklarının inşası sırasında poli-A seçiminin bir sonucu olarak gözden kaçırılır veya çok düşük bollukta bulunur ve bu nedenle izole etmek ve tespit etmek zordur. Burada, bir circRNA kütüphane inşaat protokolü kütüphane hazırlama kitleri, ön işlem seçenekleri ve çeşitli toplam RNA giriş miktarları karşılaştırılarak optimize edildi. RNase R ön işlem li ve olmayan ve değişken miktarda toplam RNA girişi (1 ila 4 g) kullanılarak, ticari olarak kullanılabilen iki tam transkripsiyon kitaplığı hazırlama kitleri test edildi. Son olarak, birden fazla doku tipleri; karaciğer, akciğer, lenf düğümü ve pankreas dahil; yanı sıra birden fazla beyin bölgeleri; beyincik dahil, inferior parietal lob, orta temporal girus, oksipital korteks, ve üstün frontal girus; doku tipleri arasında circRNA bolluğunu değerlendirmek için karşılaştırıldı. Altı farklı circRNA algılama aracı (find_circ, CIRI, Mapsplice, KNIFE, DCC ve CIRCexplorer) kullanılarak oluşturulan RNA-seq verilerinin analizi, RNase R ön işlem ve 4 μg RNA girişi ile mahsur toplam RNA kitaplık hazırlama kitinin en uygun olduğunu ortaya koymuştur. circRNA'ların en yüksek göreli sayısını belirleme yöntemi. Önceki bulgularla tutarlı olarak, beyin dokularında diğer doku tiplerine göre en yüksek circRNA zenginleştirme gözlendi.

Ticari olarak kullanılabilen evrensel kontrol RNA (UC) kullanılarak ve her ikisi de protokollerinde ribo-tükenme adımı içeren iki kitaplık hazırlama kitleri kullanılarak oluşturulan veriler ilk olarak değerlendirildi. Truseq veri kümelerinde Kapa veri kümelerine göre daha yüksek sayıda circRNA saptandı (Veri analizi iş akışı, bölüm 4), genel olarak (Şekil 1). Ribozomal RNA (rRNA) yüzdeleri daha düşük giriş miktarları için her iki kitten (1, 2 ug) veri kümeleri...

Watch this Scientific Journal Video about Identification of Circular RNAs using RNA Sequencing at JoVE.com

Identification of Circular RNAs using RNA Sequencing

Dairesel RNA'lar (circRNA'lar), transkripsiyonel düzenlemede ve proteinler arasındaki iletişimlerde rol alabilecek kodlamayan RNA'lardır. CircRNA sıralama kütüphanelerinin inşası için farklı parametrelerin değerlendirilmesinin ardından, RNase R ön arıtma ile mahsur kalan toplam RNA kitaplığı hazırlığı kullanılarak bir protokol derlenmiş ve burada sunulmuştur.

RNA Sıralaması Kullanılarak Dairesel RNA'ların Tanımlanması

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

Bu protokol, kit tipi, RNase R tedavisi, giriş miktarları ve bunların circRNA tespiti üzerindeki etkileri gibi circRNA kitaplık hazırlamanın temel yönlerini değerlendirmenin bir sonucudur. En iyi circRNA algılamasını sağlar ve kullanıcının ihtiyaçlarına bağlı olarak ayarlanabilir parametreler hakkında veri sağlar. Farklı örnek türlerinde circRNA'ların belirlenmesi, ifadelerinin dağılımını daha iyi anlamamıza yardımcı olacak ve bu moleküllerin işlevsel rolünün belirlenmesine zemin hazırlar.Bu yöntem herhangi bir toplam RNA örneğine uygulanabilir. Protokolbaşlamadan önce RNA örneklerini buzda tutmak önemlidir. Hazırlıktan önce Agilent Bioanalyzer veya TapeStation'daki RIN ve DV200 değerlerini değerlendirin ve RNA ile çalışırken uygun prosedürleri uygulayın.Toplam RNA'yı 39 mikrolitre RNase içermeyen suda dört mikrograma seyrelterek ve karıştırmak için yukarı ve aşağı borulandırarak başlayın. Ayrı bir tüpte, RNase R'yi mikrolitre başına iki birimlik bir çalışma konsantrasyonuna seyreltmek için 1x RNase R Tampon kullanın. Pipet 39 mikrolitre toplam RNA ve 10x RNase R Reaksiyon Tampon beş mikrolitre 1.5-mikrolitre reaksiyon tüpü içine, ve karıştırın.Seyreltilmiş RNase R.Then tam reaksiyon hacmine pipet ayarlamak ve 10 kez yukarı ve aşağı boru lama tarafından çözelti karıştırın altı mikrolitre ekleyin. Tüpü 37 derecelik bir su banyosuna 10 dakika yerleştirin ve tam reaksiyon hacminin su banyosuna daldırDığından emin olun. Sonra, buz üzerinde tüp yerleştirin ve hemen RNA temizleme ve konsantrasyon ile devam edin.Başlamadan önce, konsantreyi %100 etanolün 48 mililitresi ile karıştırarak RNA Yıkama Tamponu'nu hazırlayın ve arınma kolonlarını toplama tüplerine yerleştirin. Hazır olduğunuzda, RNaR işlem görmüş örneğe iki cilt RNA Bağlama Arabelleği ekleyin ve iyice karıştırın. Toplam 300 mikrolitre karışıma 150 mikrolitre %100 etanol ekleyin, tüm hacmi kolona aktarın ve 30 saniye santrifüj yapın.Akışı kaldırın ve doğrudan sütuna 400 mikrolitre RNA Hazırlık Arabelleği ekleyin. 30 saniye santrifüj ve akışı atın. Ardından, sütuna 700 mikrolitre RNA Yıkama Tamponu ekleyin, 30 saniye santrifüj yapın ve akışı atın.Yıkama Tamponbaşka 400 mikrolitre ekleyin, iki dakika için santrifüj, ve taze bir RNase-free 1.5 mililitre tüp sütun aktarın. Pipet ucunu kolon filtresinin hemen üzerinde tutarak doğrudan kolona 11 mikrolitre RNase içermeyen su ekleyin. Örnek oda sıcaklığında bir dakika oturup, ve sonra bir dakika için santrifüj edelim.1,5 mililitrelik tüpte akış olduğundan emin olun ve sütunu atın. Saflaştırılmış RNA örneği eksi 80 derecede saklanabilir veya kütüphane hazırlığı için hemen kullanılabilir. Saflaştırılmış toplam RNA'nın 10 mikrolitresini 96 kuyulu bir PCR plakalı temiz bir kuyuya aktarın.RRNA Bağlama Tampon beş mikrolitre ekleyin ve rRNA Kaldırma Mix beş mikrolitre ardından, sonra yavaşça pipet yukarı ve aşağı reaktifleri karıştırmak için. Plakayı kapatın ve önceden programlanmış ve önceden ısıtılmış bir termocycler bloğunda 68 santigrat derecede beş dakika kuluçkaya yatırın. Kuluçkadan sonra, plaka bir dakika oda sıcaklığında oturalım.Mührü plakadan çıkarın ve numuneye 35 mikrolitre girdaplı oda sıcaklığında rRNA kaldırma boncukları ekleyin. Pipeti 45 mikrolitreye, pipetleri de iyice karıştırmak için 10 ila 20 kez yukarı ve aşağı ayarlayın. Plaka bir dakika oda sıcaklığında oturalım.Plakayı manyetik bir standa aktarın ve bir dakika veya çözelti temize çıkana kadar orada kuluçkaya yatırın. Supernatant'ı plakadaki yeni kuyuya aktarın. Daha sonra plakayı manyetik standdan plaka standına aktarın.Vortex RNA iyi dağılmış kadar boncuk temizleme ve örnek için boncuk 99 mikrolitre ekleyin. Pipet yukarı ve aşağı karıştırmak için, ve oda sıcaklığında 10 dakika boyunca tabak kuluçka. Plakayı manyetik standa aktarın ve beş dakika veya çözelti temize çıkana kadar orada bırakın, sonra tüm supernatant'ı kuyudan çıkarın ve atın.Plaka hala manyetik stand üzerinde yken, boncukları bozmadan kuyuya 200 mikrolitre taze hazırlanmış %80 etanol ekleyin. 30 saniye boyunca kuyuda etanol bırakın, sonra çıkarın ve atın. Kuyuya 11 mikrolitre Elütion Tampon ekleyin ve karıştırmak için yukarı ve aşağı pipet.Plakayı oda sıcaklığında iki dakika kuluçkaya yatırın, sonra manyetik standa geri aktarın ve çözelti temize çıkana kadar orada tutun. Süpernatantın 8,5 mikrolitresini aynı plakaüzerindeki yeni bir kuyuya aktarın ve numune içeren her kuyuya 8,5 mikrolitre Elute, Primer, Fragment High Mix ekleyin. Pipet yukarı ve aşağı 10 kez iyice karıştırmak için, plaka mühür ve 94 santigrat derece önceden ısıtılmış bir termocycler sekiz dakika kuluçka.Numuneyi dört dereceye kadar soğutun, plakayı termocycler'dan çıkarın ve kısa bir süre santrifüj edin. Bu protokol için TruSeq ve KAPA olmak üzere iki kütüphane hazırlama kitleri değerlendirildi. Genel olarak, TruSeq kiti ni kullanırken daha yüksek sayıda dairesel RNA ve daha düşük ribozomal RNA yüzdesi saptandı, bu yüzden daha fazla deney için TruSeq seçildi.RNase R ön tedavisinin önemi, önceden tedavi edilmiş ve tedavi edilmeyen kütüphanelerden elde edilen veriler karşılaştırılarak değerlendirildi. Beklendiği gibi, önceden tedavi edilmeyen kütüphanelere kıyasla, daha yüksek sayıda dairesel RNA tutarlı bir şekilde önceden tedavi edilmemiş kütüphanelerde tanımlanmıştır. Giriş RNA miktarı dairesel RNA'ların daha yüksek bir çeşitliliğini tespit etmek için optimize edildi.Kütüphaneler bir, iki ve dört mikrogram giriş RNA kullanılarak hazırlandı ve dört mikrogram toplam RNA kullanırken dairesel RNA türlerinin en yüksek çeşitliliği gözlendi. Optimize edilmiş protokol, dört sağlıklı donörden beş beyin bölgesinde ve altı sağlıklı donörün diğer doku tipleri için dairesel RNA bolluklarını karşılaştırmak için kullanılmıştır. Genel olarak, dairesel RNase daha yüksek bir bolluk diğer doku tiplerine göre beyinde gözlendi.RNA ile çalışırken eldiven takmak, protokole başlamadan önce tezgah ve pipetleri RNase mendilleriyle veya spreyle iyice temizlemek ve RNA'yı önceden buzda tutmak da dahil olmak üzere uygun prosedürleri izlemeyi unutmamak önemlidir. Bu prosedürü takiben, tanımlanan circRNA kavşaklarının Sanger sıralanması gibi ortogonal doğrulama yapılabilir. Yeni circRNA'ların keşfi ve onların varlığına dair daha fazla kanıt, biyolojik işlevlerini keşfetmek için yeni bir araştırma alanı ortaya çıkarıyor.

identification-of-circular-rnas-using-rna-sequencing

Research

JoVE Journal

Genetics

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

RNA-RNA Etkileşmelerini Biyotinlenmiş Psooren Kullanarak Küresel Olarak Haritalama

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Genetik

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

Metabolik etiketleme ve Transfer RNA Macroarrays kullanarak profil oluşturma

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

Retrospektif mikroRNA sıralama: Parafin gömülü RNA Formalin sabit örnekler kullanarak Tamamlayıcı DNA Kütüphane hazırlık Protokolü

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Nadiren de olsa algılama hatası düzeltildi DNA ve RNA sıralama

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

Gen düzenleyici dizileri dairesel kromozom Conformation yeni nesil sıralama kullanarak yüksek üretilen iş tanımlaması yakalama (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since 2007. These studies have confirmed that lncRNAs are altered in almost all diseases. However, studying the functional roles for lncRNAs in the context of disease remains difficult due to the lack of protein products, tissue-specific expression, low expression levels, complexities in splice forms, and lack of conservation among species. Given the species-specific expression, lncRNA studies are often restricted to human research contexts when studying disease processes. Since lncRNAs function at the molecular level, one way to dissect lncRNA biology is to either remove the lncRNA or overexpress the lncRNA and measure cellular effects. In this article, a written and visualized protocol to overexpress lncRNAs in vitro is presented. As a representative experiment, an lncRNA associated with inflammatory bowel disease, Interferon Gamma Antisense 1 (IFNG-AS1), is shown to be overexpressed in a Jurkat T-cell model. To accomplish this, the activating clustered regularly interspaced short palindromic repeats (CRISPR) technique is used to enable overexpression at the endogenous genomic loci. The activating CRISPR technique targets a set of transcription factors to the transcriptional start site of a gene, enabling a robust overexpression of multiple lncRNA splice forms. This procedure will be broken down into three steps, namely (i) guide RNA (gRNA) design and vector construction, (ii) virus generation and transduction, and (iii) colony screening for overexpression. For this representative experiment, a greater than 20-fold enhancement in IFNG-AS1 in Jurkat T cells was observed.

Traditional cDNA-based overexpression techniques have a limited applicability for the overexpression of long noncoding RNAs due to their multiple splice forms with potential functionality. This review reports a protocol using CRISPR technology to overexpress multiple splice variants of a long noncoding RNA.

Gene aktive CRISPR kullanarak uzun kodlamayan RNA'ların overexpressing

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Nanopore sıralamayı kullanarak tüm kan mRNA sıralaması

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Insan pankreatik Izletinin tek hücreli RNA sıralaması ve Analizi

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA 'nın ayak Izleri tanımlaması: proteinli kompleksleri RNA Immunoprecipitation ile tandem sonra sıralamayı (RIPiT-seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

Improving Small RNA-seq: Less Bias and Better Detection of 2&#039;-O-Methyl RNAs

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

Küçük RNA-seq iyileştirilmesi: Daha Az Önyargı ve 2'-O-Metil RNA'ların Daha İyi Tespiti

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...