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

Vi er takknemlige for banner Søn Health Research Institute Brain and Body donasjon program (BBDP) av Sun City, Arizona for levering av menneskelige hjernevev. Den BBDP har blitt støttet av National Institute of nevrologiske lidelser og hjerneslag (U24 NS072026 National Brain og vev Resource for Parkinsons sykdom og relaterte lidelser), National Institute on aging (P30AG19610 Arizona Alzheimers sykdom Core Center), Arizona Department of Health Services (kontrakt 211002, Arizona Alzheimers Research Center), Arizona Biomedical Research Commission (kontrakter 4001, 0011, 05-901 og 1001 til Arizona Parkinson ' s Disease Consortium) og Michael J. Fox Foundation for Parkinsons forskning27. Denne studien ble også støttet av DHS og staten Arizona (ADHS Grant # ADHS14-052688). Vi takker også Andrea Schmitt (banner Research) og Cynthia Lechuga (TGen) for Administrativ støtte.

Denne forskningen er utført i samsvar med alle institusjonelle, nasjonale og internasjonale retningslinjer for menneskelig velferd. Brain vev ble innhentet fra banner Søn Health Research Institute Brain og Body donasjon program i Sun City, AZ. Driften av Brain and Body donasjon program er godkjent av Western institusjonelle Review Board (WIRB protokollen #20120821). Alle eller deres juridiske representanter undertegnet informert samtykke. Kommersielle (ikke-hjerne) biospecimens ble kjøpt fra Proteogenex.
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<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. 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I denne studien, to kommersielt tilgjengelige bibliotek forberedelse kits, pre-behandling alternativer, og innspill RNA beløpene ble testet for å optimalisere en circRNA berikelse protokoll for bygging av circRNA sekvensering biblioteker. Basert på vurderingen til denne studien, er en rekke viktige aspekter og kritiske trinn for å opprette circRNA sekvensering biblioteker åpenbare. Vår evaluering bekrefter nytten av RNase R pre-behandling, som gjenspeiles av det økte antallet circRNAs oppdaget. Generelt sett ble d...

Sirkulær RNAs (CircRNAs) er endogene, ikke-koding RNAs som har fått oppmerksomhet gitt deres gjennomgående uttrykk i eukaryote transcriptome1,2,3. De dannes når exoner back-skjøte til hverandre og dermed ble i utgangspunktet anses å være skjøting gjenstander4,5. Men nyere studier har vist at circRNAs utstillingen celle type, vev, og utviklingsmessige scenen spesifikke uttrykk3,6 og er evolutionarily bevart2,3. Videre er de involvert i mekling av protein-protein interaksjoner7, mikro-RNA (miRNA) binding3,8,9,10, og regulering av foreldrenes genet transkripsjon11.
I klassisk RNA sekvensering (RNA-SEQ), circRNAs kan være helt tapt under biblioteket konstruksjon som et resultat av Poly-et utvalg for mRNAs eller kan være vanskelig å isolere gitt sin lave overflod. Men nylig circRNA karakterisering studier har innarbeidet en pre-Treatment trinn ved hjelp RNase R for å berike for circRNAs2,12,13. RNase R er en exoribonuclease som fordøyer lineær RNAs, og etterlater sirkulære RNA-strukturer. CircRNA berikelse protokoller ble optimalisert ved å generere og sammenligne data fra to kommersielt tilgjengelige hele transcriptome biblioteket konstruksjon kits, med og uten en RNase R pre-Treatment trinn, og ved hjelp av varierende mengder av total RNA input (1 til 4 μg). Den optimaliserte protokollen ble neste brukt til å evaluere overflod av circRNAs over fem forskjellige hjernen regioner (lillehjernen [BC], mindreverdig parietal flik [IP], midt Temporal gyrus [MG], occipital cortex [OC] og overlegen frontal gyrus [SF]) og fire andre vev typer (lever [LV], lunge [LU], lymfeknuter [LN] og bukspyttkjertel [PA]). RNA-SEQ bibliotekene ble sammenkoblet slutten sekvensert og data ble analysert ved hjelp av seks forskjellige circRNA prediksjon algoritmer: find_circ3, CIRI14, Mapsplice15, Knife16, DCC17, og CIRCexplorer18. Basert på vår analyse ble det høyeste antallet unike circRNAs oppdaget ved bruk av et strandet total RNA-biblioteks Forberedelses sett med RNase R forbehandling og 4 μg total inngang RNA. Den optimaliserte protokollen er beskrevet her. Som tidligere rapportert19,20, den høyeste berikelse av circRNAs ble observert i hjernen i forhold til andre typer vev.

<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

Circular RNAs (circRNAs) er en klasse av ikke-koding RNAs involvert i funksjoner, inkludert Micro-RNA (miRNA) regulering, formidling av protein-protein interaksjoner, og regulering av foreldrenes genet transkripsjon. I klassisk neste generasjon RNA sekvensering (RNA-SEQ), circRNAs er vanligvis oversett som følge av Poly-et utvalg under byggingen av mRNA biblioteker, eller finnes ved svært lav overflod, og er derfor vanskelig å isolere og oppdage. Her, en circRNA biblioteket konstruksjon protokollen ble optimalisert ved å sammenligne biblioteket forberedelse kits, forbehandling alternativer og ulike totale RNA input beløp. To kommersielt tilgjengelige hele transcriptome bibliotek Forberedelses sett, med og uten RNase R forbehandling, og ved hjelp av variable mengder totalt RNA-inngang (1 til 4 μg), ble testet. Til slutt, flere vevstyper; inkludert lever, lunge, lymfe node, og bukspyttkjertel; så vel som flere hjerneregioner; inkludert lillehjernen, mindreverdig parietal flik, midt Temporal gyrus, occipital cortex, og overlegen frontal gyrus; sammenlignet med å evaluere circRNA overflod på tvers av vevstyper. Analyse av de genererte RNA-SEQ-dataene ved hjelp av seks forskjellige circRNA gjenkjenningsverktøy (find_circ, CIRI, Mapsplice, KNIFE, DCC og CIRCexplorer) avslørte at en strandet total RNA-biblioteks Forberedelses sett med RNase R forbehandling og 4 μg RNA-inngang er den optimale metode for å identifisere det høyeste relative antallet circRNAs. I samsvar med tidligere funn, den høyeste berikelse av circRNAs ble observert i hjernen vev i forhold til andre typer vev.

Data generert ved hjelp av en kommersielt tilgjengelig universell kontroll RNA (UC) og bruk av to biblioteks Forberedelses sett, som begge inkluderer et Ribo-trinn i protokollene, ble først vurdert. Ved hjelp av en analytisk arbeidsflyt (arbeidsflyt for data analyse, del 4), samlet, ble et høyere antall circRNAs oppdaget i TruSeq datasett sammenlignet med de kapa de (figur 1). Selv om ribosomal RNA (rRNA) prosenter var under 5% i datasett fra begge settene for lavere input beløp (1, 2 UG)...

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

Identification of Circular RNAs using RNA Sequencing

Sirkulær RNAs (circRNAs) er ikke-koding RNAs som kan ha roller i transcriptional regulering og formidling interaksjoner mellom proteiner. Etter vurdering av ulike parametre for bygging av circRNA sekvensering biblioteker, en protokoll ble kompilert utnytte strandet total RNA biblioteket forberedelse med RNase R pre-behandling og er presentert her.

Identifikasjon av sirkulær RNAs ved hjelp av RNA-sekvensering

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

Denne protokollen er et resultat av å evaluere viktige aspekter ved circRNA bibliotek forberedelse, for eksempel kit type, RNase R behandling, inngangsbeløp og deres innvirkning på circRNA deteksjon. Det muliggjør optimal circRNA-deteksjon og gir data om justerbare parametere avhengig av brukerens behov. Identifisering av circRNAs i forskjellige utvalgstyper vil hjelpe oss med å bedre forstå fordelingen av uttrykket deres og setter scenen for å avgrense de funksjonelle rollen til disse molekylene.Denne metoden kan brukes på alle totale RNA-eksempler. Det er viktig å holde RNA-prøver på is før protokollen startes. Vurder RIN- og DV200-verdiene på Agilent Bioanalyzer eller TapeStation før forhåndsforberedelse, og følg passende prosedyrer når du arbeider med RNA.Begynn med å fortynne totalt RNA til fire mikrogram i 39 mikroliter RNase-fritt vann og pipetter det opp og ned for å blande. I et separat rør bruker du 1x RNase R Buffer til å fortynne RNase R til en arbeidskonsentrasjon på to enheter per mikroliter. Pipette 39 mikroliter av totalt RNA og fem mikroliter på 10x RNase R reaksjonsbuffer i et reaksjonsrør på 1,5 mikroliter, og bland.Tilsett seks mikroliter av den fortynnede RNase R.Juster deretter pipetten til fullt reaksjonsvolum, og bland løsningen ved å pipetter opp og ned 10 ganger. Plasser røret i et 37-graders Celsius vannbad i 10 minutter, og sørg for at hele reaksjonsvolumet er nedsenket i vannbadet. Deretter plasserer du røret på is, og fortsett umiddelbart med RNA-opprydding og konsentrasjon.Før du starter, klargjør RNA Wash Buffer ved å blande konsentratet med 48 milliliter med 100% etanol, og plasser rensekolonner i oppsamlingsrør. Når du er klar, legger du til to binderingsbuffer for RNA i RNase R-behandlet prøve og blander godt. Tilsett 150 mikroliter med 100% etanol til blandingen for totalt 300 mikroliter, overfør hele volumet til kolonnen og sentrifuge i 30 sekunder.Fjern flyten gjennom, og legg til 400 mikroliter RNA Prep Buffer direkte i kolonnen. Sentrifuge i 30 sekunder, og kast strømmen gjennom. Deretter legger du til 700 mikroliter RNA-vaskebuffer i kolonnen, sentrifuge i 30 sekunder og kast strømmen gjennom.Legg til ytterligere 400 mikroliter av vaskebufferen, sentrifuge i to minutter, og overfør kolonnen til et friskt RNase-fritt 1,5-milliliter rør. Tilsett forsiktig 11 mikroliter RNase-fritt vann direkte til kolonnen ved å holde pipettespissen rett over kolonnefilteret. La prøven sitte i ett minutt ved romtemperatur, og sentrifuge den i ett minutt.Kontroller at det er gjennomstrømning i 1,5-milliliter røret, og kast kolonnen. Den rensede RNA-prøven kan lagres ved minus 80 grader Celsius eller brukes umiddelbart til bibliotekforberedelse. Overfør 10 mikroliter av den rensede totale RNA til en ren brønn i en 96-brønns PCR-plate.Tilsett fem mikroliter rRNA Binding Buffer etterfulgt av fem mikroliter rRNA Removal Mix, og pipette deretter forsiktig opp og ned for å blande reagensene. Forsegle platen, og inkuber den i fem minutter ved 68 grader Celsius på en forhåndsprogrammert og forvarmet termocyclerblokk. Etter inkubasjonen, la platen sitte ved romtemperatur i ett minutt.Fjern forseglingen fra platen, og tilsett 35 mikroliter av vortexed romtemperatur rRNA fjerning perler til prøven. Juster pipetten til 45 mikroliter, og pipetten opp og ned 10 til 20 ganger for å blandes grundig. La platen sitte ved romtemperatur i ett minutt.Overfør platen til et magnetisk stativ, og inkuber den der i ett minutt eller til løsningen klargjør. Overfør det overnaturlige til ny brønn på tallerkenen. Overfør deretter platen fra magnetstativet til platestativet.Vortex RNA opprydding perler til de er godt dispergert, og tilsett 99 mikroliter perler til prøven. Pipetten opp og ned for å blande, og inkubere platen ved romtemperatur i 10 minutter. Overfør platen til magnetstativet, og la den stå der i fem minutter eller til løsningen klarner, og fjern og kast alle overnaturlige fra brønnen.Med platen fortsatt på magnetstativet, legg til 200 mikroliter med nylaget 80% etanol til brønnen uten å forstyrre perlene. La etanol i brønnen stå i 30 sekunder, fjern og kast den. Tilsett 11 mikroliter elutionbuffer til brønnen, og pipetter den opp og ned for å blande.Inkuber platen ved romtemperatur i to minutter, og overfør den deretter tilbake til magnetstativet, og hold den der til løsningen er klar. Overfør 8,5 mikroliter av supernatanten til en ny brønn på samme plate, og tilsett 8,5 mikroliter Elute, Primer, Fragment High Mix til hver brønn som inneholder en prøve. Pipetten opp og ned 10 ganger for å blande grundig, forsegle platen og inkubere den i åtte minutter i en termocycler forvarmet til 94 grader Celsius.Avkjøl prøven til fire grader Celsius, fjern platen fra termocycleren, og sentrifuger den kort. To bibliotekforberedelsessett, TruSeq og KAPA, ble vurdert for denne protokollen. Samlet sett ble det påvist et høyere antall sirkulære RNA-er og lavere prosentandel ribosomal RNA ved bruk av TruSeq-settet, så TruSeq ble valgt for videre eksperimenter.Betydningen av RNase R-forbehandling ble evaluert ved å sammenligne dataene som genereres fra forbehandlede og ikke-forhåndsbehandlede biblioteker. Som forventet ble et høyere antall sirkulære RNA-er konsekvent identifisert i de forbehandlede bibliotekene sammenlignet med ikke-forhåndsbehandlede. Mengden input RNA ble optimalisert for å oppdage et høyere mangfold av sirkulære RNAAer.Bibliotekene ble utarbeidet ved hjelp av ett, to og fire mikrogram input RNA, og det høyeste mangfoldet av sirkulære RNA-arter ble observert ved bruk av fire mikrogram totalt RNA. Den optimaliserte protokollen ble brukt til å sammenligne sirkulære RNA overflod i fem hjerneregioner fra fire friske givere og for andre vevstyper fra seks friske givere. Samlet sett ble en høyere overflod av sirkulære RNase observert i hjernen sammenlignet med andre vevstyper.Det er viktig å huske å følge passende prosedyrer når du arbeider med RNA, inkludert bruk av hansker, grundig rengjøring av benken og pipetter med RNase kluter eller spray før du begynner protokollen, og holde RNA på is på forhånd. Etter denne prosedyren kan ortogonal validering som Sanger sekvensering av identifiserte circRNA-veikryss utføres. Oppdagelsen av nye circRNAs og ytterligere bevis på deres tilstedeværelse skjærer ut et nytt forskningsområde for å utforske deres biologiske funksjoner.

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.

Kartlegging av RNA-RNA-interaksjoner globalt ved bruk av biotinylert 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 ...

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.

Metabolsk merking og profilering av overføring RNAs bruker 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 ...

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.

Retrospektiv MicroRNA sekvensering: Komplementære DNA biblioteket forberedelse protokollen bruker Formalin-fast parafin-embedded RNA prøver

&#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.

Sjelden hendelse gjenkjenning feil-korrigert DNA og RNA sekvensering

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.

Høy gjennomstrømming identifikasjon av regulatoriske gensekvenser med neste generasjons sekvensering av runde kromosom konformasjon fange (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.

Overexpressing lang Noncoding RNAs med aktivering av Gene 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 ...

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.

Sekvensering av mRNA fra hele Blood bruke Nanopore sekvensering

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.

Single-Cell RNA sekvensering og analyse av Human bukspyttkjertelen holmer

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).

Identifisering av fotavtrykk av RNA: protein komplekser via RNA-Immunutfelling i tandem etterfulgt av sekvensering (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.

Forbedre små RNA-SEQ: mindre bias og bedre påvisning av 2 '-O-metyl 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 ...