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

アリゾナ州サンシティのバナーサン健康研究所脳体寄付プログラム(BBDP)に感謝しています。BBDPは、国立神経疾患・脳卒中研究所(U24 NS072026パーキンソン病および関連障害のための国立脳・組織資源)、国立加齢研究所(P30AG19610アリゾナアルツハイマー病コアセンター)、アリゾナ州保健サービス省(契約211002、 アリゾナ・アルツハイマー研究センター)、アリゾナ生物医学研究委員会(アリゾナ・パーキンソン病コンソーシアムに4001、0011、05-901、1001を契約)、マイケル・J。フォックスパーキンソン病研究財団27.この研究はまた、DHSとアリゾナ州(ADHS14-052688)によって支持されました。また、アンドレア・シュミット(バナー・リサーチ)とシンシア・レチュガ(TGen)の行政支援に感謝します。

本研究は、人間の福祉に関するすべての制度、国内、国際的なガイドラインに準拠して行われています。脳組織は、AZのサンシティにあるバナーサン健康研究所脳体寄付プログラムから得られました。脳と身体の寄付プログラムの運営は、西部機関審査委員会(WIRBプロトコル#20120821)によって承認されています。すべてのサブジェクトまたはその法的代理人がインフォームド コンセントに署名?...

<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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viroids+are+single-stranded+covalently+closed+circular+RNA+molecules+existing+as+highly+base-paired+rod-like+structures.">Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures.</a> Proceedings of the National Academy of Sciences of the United States of America. 73 (11), 3852-3856 (1976).</li><li>Nigro, J. 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=Scrambled+exons.">Scrambled exons.</a> Cell. 64 (3), 607-613 (1991).</li><li>Salzman, J., Chen, R. E., Olsen, M. N., Wang, P. L., 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=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. 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=miRNA-dependent+gene+silencing+involving+Ago2-mediated+cleavage+of+a+circular+antisense+RNA.">miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA.</a> The EMBO Journal. 30 (21), 4414-4422 (2011).</li><li>Hansen, T. 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=Natural+RNA+circles+function+as+efficient+microRNA+sponges.">Natural RNA circles function as efficient microRNA sponges.</a> Nature. 495 (7441), 384-388 (2013).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exon-intron+circular+RNAs+regulate+transcription+in+the+nucleus.">Exon-intron circular RNAs regulate transcription in the nucleus.</a> Nature Structural &amp; Molecular Biology. 22 (3), 256-264 (2015).</li><li>Tan, W. 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. G., 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=Arizona+study+of+aging+and+neurodegenerative+disorders+and+brain+and+body+donation+program.">Arizona study of aging and neurodegenerative disorders and brain and body donation program.</a> Neuropathology. 35 (4), 354-389 (2015).</li></ol>

本研究では、2つの市販のライブラリー調製キット、前処理オプション、および入力RNA量を試験し、circRNAシーケンシングライブラリの構築のためのcircRNA濃縮プロトコルを最適化した。この研究の評価に基づいて、circRNAシーケンシングライブラリを作成する際の重要な側面と重要なステップの数が明らかです。我々の評価は、検出されたcircRNAの数の増加によって反映されるRNase R前処理の有用?...

円形RNA(CircRNA)は、真核生物転写1、2、3における広範な発現を考えると注目を集めている内因性の非符号化RNAである。それらは、互いに逆スプライスを取り除いたときに形成され、したがって、最初はアーティファクト4、5をスプライシングしていると考えられていました。しかしながら、最近の研究では、circRNAが細胞型、組織、および発達段階特異的発現3、6を示し、進化的に保存されている2、3が実証されている。さらに、それらは、タンパク質間相互作用7のメディエーションに関与している7、マイクロRNA(miRNA)結合3、8、9、10、および親遺伝子転写11の調節である。
古典的なRNAシーケンシング(RNA-seq)では、mRNAのポリA選択の結果としてライブラリの構築中にcircRNAが完全に失われたり、その低い存在量を考えると分離が困難な場合があります。しかしながら、最近のcircRNA特性評価研究は、CircRNA2、12、13を濃縮するためにRNase Rを用いた前処理工程を組み込んだ。RNase Rは、円形RNA構造の後ろに残して、線形RNAを消化するエキボリボヌクレアーゼです。CircRNAエンリッチメントプロトコルは、RNase R前処理ステップの有無にかかわらず、市販されている2つの全トランストランスクリプトームライブラリ構築キットからデータを生成および比較し、さまざまな量の合計RNA入力(1〜4μg)を使用することによって最適化されました。最適化されたプロトコルは、次に5つの異なる脳領域(小脳[BC]、下頭頭葉[IP]、中側頭蓋[MG]、後頭前皮質[OC]および上前頭回[SF])および4つの他の組織タイプ(肝臓[LV]、肺[LU]、リンパ節[LN]および膵臓[PA])にわたるcircRNAの豊富さを評価するために使用された。RNA-seqライブラリをペアにエンドシーケンスし、データを6つの異なるcircRNA予測アルゴリズムを使用して分析した:find_circ 3、CIRI14、マッププライス15、KNIFE16、DCC17、およびCIRCexplorer18。我々の分析に基づいて、RNase R前処理および4μgの総入力RNAとの孤立した総RNAライブラリー調製キットを使用した場合、最も多くのユニークなcircRNAが検出されました。ここでは、最適化されたプロトコルについて説明します。以前に報告された19、20として、他の組織タイプと比較して脳内で最も高い濃縮が観察された。

<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

円形RNA(circRNA)は、マイクロRNA(miRNA)調節、タンパク質間相互作用のメディエーション、親遺伝子転写の調節などの機能に関与する非コードRNAのクラスです。古典的な次世代RNAシーケンシング(RNA-seq)では、circRNAは通常、mRNAライブラリの構築中にポリA選択の結果として見落とされるか、または非常に低い存在量で発見され、したがって分離および検出することは困難です。ここで、circRNAライブラリ構築プロトコルは、ライブラリ調製キット、前処理オプションおよび各種全RNA入力量を比較することによって最適化された。RNase R前処理の有無にかかわらず、市販の2つの全トランストランスクリプトームライブラリ調製キット、および可変量の合計RNA入力(1〜4μg)を使用して、試験した。最後に、複数の組織タイプ;肝臓、肺、リンパ節、および膵臓を含む;だけでなく、複数の脳領域;小脳、下頭頂葉、中側頭回、後頭皮質、および上頭前頭回を含む;組織型全体にわたるcircRNAの存在量を評価するために比較した。6つの異なるcircRNA検出ツール(find_circ、CIRI、マッププライス、ナイフ、DCC、およびCIRCexplorer)を用いた生成されたRNA-seqデータの分析により、RNase R前処理と4μg RNA入力を備えた孤立した合計RNAライブラリ調製キットが最適であることを明らかにした。circRNA の最大相対数を識別するためのメソッド。以前の知見と一致して、circRNAの最も高い濃縮は、他の組織タイプと比較して脳組織で観察された。

市販のユニバーサルコントロールRNA(UC)を用いて生成されたデータと、プロトコルにリボ枯渇ステップを含む2つのライブラリ調製キットを使用して、最初に評価された。分析ワークフロー (データ分析ワークフロー、セクション 4) を使用して、全体的に、Kapa データセットと比較して TruSeq データセットでより多くの circRNA が検出されました (図 1)。リボソームRNA(rRNA)の?...

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

Identification of Circular RNAs using RNA Sequencing

円形RNA(circRNA)は、タンパク質間の転写調節および媒介相互作用に役割を有し得る非コードRNAである。circRNAシーケンシングライブラリの構築のための異なるパラメータの評価に続いて、RNase R前処理を用いた立ち往生した全RNAライブラリ調製を利用してプロトコルをコンパイルし、ここで提示する。

RNA シーケンシングを使用した円形 RNA の同定

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

このプロトコルは、キットタイプ、RNase R処理、入力量、およびcircRNA検出への影響など、circRNAライブラリ調製の主要な側面を評価した結果です。それは最適なサークRNAの検出を可能にし、ユーザーの必要性に応じて調節可能な変数のデータを提供する。異なるサンプルタイプのcircRNAを同定することは、その発現の分布をよりよく理解するのに役立ち、これらの分子の機能的役割を解明する段階を設定します。この方法は、任意の合計RNAサンプルに適用することができる。プロトコルを開始する前に、RNAサンプルを氷の上に保管しておくことが重要です。準備前にアジレントバイオアナライザまたはテープステーションでRINとDV200の値を評価し、RNAを使用する場合は適切な手順に従ってください。まず、合計RNAをRNaseフリーウォーターの39マイクロリットルで4マイクログラムに希釈し、上下にピペットして混合します。別のチューブでは、1x RNase Rバッファーを使用して、RNase Rをマイクロリットル当たり2単位の作業濃度に希釈します。ピペット39マイクロリットルの全RNAと5マイクロリットルの10倍RNase R反応バッファーを1.5マイクロリットル反応チューブに混合し、混合します。希釈したRNase R.の6マイクロリットルを加え、ピペットを完全な反応量に調整し、上下にピペットを10回混ぜます。チューブを摂氏37度の水浴に10分間入れ、完全な反応量が水浴に浸かっていることを確認します。次いで、チューブを氷の上に置き、すぐにRNAのクリーンアップと濃縮を進めます。始める前に、濃縮物に100%エタノールの48ミリリットルを混合してRNAウォッシュバッファーを調製し、精製カラムを回収管に入れます。準備ができたら、RNase R処理サンプルに2つの量のRNA結合バッファーを加え、よく混ぜます。100%エタノールの150マイクロリットルを混合物に加え、合計300マイクロリットル、全容をカラムに移し、遠心分離機を30秒間加えます。流れを取り除き、400マイクロリットルのRNA準備バッファーをカラムに直接加えます。遠心分離機を30秒間、流れを捨てます。その後、カラムに700マイクロリットルのRNAウォッシュバッファーを加え、遠心分離機を30秒間加え、流れを通して廃棄します。さらに400マイクロリットルのウォッシュバッファー、遠心分離機を2分間加え、カラムを新鮮なRNaseフリーの1.5ミリリットルチューブに移します。柱フィルターの真上にピペットチップを持って、RNaseフリー水11マイクロリットルを柱に直接慎重に加えます。サンプルを室温で1分間座らせてから、1分間遠心分離します。1.5ミリリットルのチューブに流れが通っていることを確認し、カラムを捨てます。精製されたRNAサンプルは、マイナス80°Cで保存することも、ライブラリの調製にすぐに使用することもできます。精製した全RNAの10マイクロリットルを96ウェルPCRプレート内のクリーンウェルに移します。rRNA結合バッファーの5マイクロリットルを追加し、rRNA除去ミックスの5マイクロリットルを追加し、試薬を混合するために上下にピペットを穏やかにピペット。プレートを密封し、事前にプログラムされ、予熱されたサーモサイクラーブロックで摂氏68度で5分間インキュベートします。インキュベーションの後、プレートを室温で1分間座らせます。プレートからシールを取り出し、35マイクロリットルの渦中室温rRNA除去ビーズをサンプルに加えます。ピペットを45マイクロリットルに調整し、ピペットを10~20回上下に調整して完全に混合します。プレートを室温で1分間座らせます。プレートを磁気スタンドに移し、1分間、または溶液がクリアされるまでインキュベートします。上清をプレート上の新しい井戸に移します。次に、磁気スタンドからプレートスタンドにプレートを移します。ボルテックスRNAクリーンアップビーズは、それらが十分に分散されるまで、サンプルにビーズの99マイクロリットルを追加します。ピペットを上下に混ぜ合わせ、室温で10分間インキュベートします。プレートを磁気スタンドに移し、5分間放置するか、溶液がクリアするまで放置し、井戸からすべての上澄み物を取り出して捨てます。磁気スタンドにプレートを残したまま、ビーズを破壊することなく、準備したての80%エタノールを200マイクロリットルのウェルに加えます。エタノールを井戸に30秒間放置し、取り除いて捨てます。ウェルに溶出バッファーの 11 マイクロリットルを追加し、ピペットを上下に混合します。プレートを室温で2分間インキュベートし、磁気スタンドに戻し、溶液がクリアされるまでそこに保管します。8.5マイクロリットルの上清を同じプレート上の新しいウェルに移し、サンプルを含む各ウェルに8.5マイクロリットルのエルテ、プライマー、フラグメントハイミックスを加えます。ピペットを10回上下して完全に混ぜ、プレートを密封し、摂氏94度に予熱したサーモサイクラーで8分間インキュベートします。サンプルを摂氏4度に冷却し、サーモサイクラーからプレートを取り出し、遠心分離機を短くします。このプロトコルについて、TruSeqとKAPAの2つのライブラリ調製キットを評価しました。全体として、TruSeqキットを使用した場合、より多くの円形RNAおよび低い割合のリボソームRNAが検出されたので、TruSeqはさらなる実験のために選択された。RNase R前処理の意義は、前処理されたライブラリと非処理されたライブラリから生成されたデータを比較することによって評価した。予想通り、未治療ライブラリと比較して、より多くの円形RNAが一貫して前処理されたライブラリで同定された。入力RNAの量は、より高い多様性の円形RNAを検出するために最適化されました。ライブラリーは、1マイクログラム、2マイクログラム、4マイクログラムの入力RNAを用いて作成し、4マイクログラムの全RNAを使用した場合には、円形RNA種の最も高い多様性が観察された。最適化されたプロトコルは、4つの健康なドナーからの5つの脳領域および6人の健康なドナーからの他の組織タイプの円形RNAの豊富さを比較するために使用された。全体として、他の組織タイプと比較して、脳内で円形RNaseの存在量が高いことが観察された。手袋を着用する、RNaseワイプまたはスプレーでベンチとピペットを徹底的に洗浄する、事前にRNAを氷の上に保つなど、RNAを扱う場合は、適切な手順に従うことを忘れないでください。この手順に従って、同定されたcircRNA接合部のサンガーシーケンシングなどの直交検証を行うことができる。新しいcircRNAの発見とその存在のさらなる証拠は、その生物学的機能を探求するための新しい研究分野を切り出す。

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.

ビオチン化ソラレンを用いたRNA-RNA相互作用のマッピング

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 ...

遺伝学

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.

代謝ラベリングと Macroarrays を使用して転移 Rna のプロファイリング

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.

ホルマリン固定パラフィン包埋 RNA 試料を用いた回顧マイクロ Rna シーケンス: 相補的な DNA ライブラリの準備のプロトコル

&#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.

エラー修正 DNA ・ RNA シーケンスを用いた珍しいイベント検出

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.

円の染色体構造の次世代シーケンシングによる遺伝子制御配列の高スループット識別キャプチャ (4 C 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.

CRISPR の遺伝子活性化を使用して長い Rna を過剰発現

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.

ナノポアシーケンシングを用いた全血からの mRNA の配列決定

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.

ヒト膵島の単一細胞RNAシーケンシングと解析

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).

ランデムにおけるRNA免疫沈殿によるRNAタンパク質複合体の足跡の同定、シーケンシング(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.

小型RNA-seqの改善:バイアスの減少と2'-O-メチルRNAの検出の改善

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 ...