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

We zijn dankbaar voor de banner Sun Health Research Institute Brain and Body donatieprogramma (BBDP) van Sun City, Arizona voor de verstrekking van menselijke hersenweefsels. De BBDP is gesteund door het National Institute of neurologische aandoeningen en beroerte (U24 NS072026 nationale hersenen en weefsel resource voor de ziekte van Parkinson en verwante aandoeningen), het Nationaal Instituut voor veroudering (P30AG19610 Arizona Alzheimer Disease core Center), de Arizona Department of Health Services (contract 211002, Arizona Alzheimer Research Center), de Arizona Biomedical Research Commission (contracten 4001, 0011, 05-901 en 1001 aan het Arizona Parkinson disease Consortium) en de Michael J. Fox Foundation voor onderzoek van Parkinson27. Deze studie werd ook gesteund door de DHS en de staat Arizona (ADHS Grant # ADHS14-052688). We bedanken ook Andrea Schmitt (banner Research) en Cynthia lechuga (TGen) voor administratieve ondersteuning.

Dit onderzoek is uitgevoerd in overeenstemming met alle institutionele, nationale en internationale richtlijnen voor het welzijn van de mens. Hersenen weefsels werden verkregen uit de banner Sun Health Research Institute hersenen en lichaam donatieprogramma in Sun City, AZ. De operaties van het brein-en lichaams donatieprogramma zijn goedgekeurd door de Western Institutional Review Board (WIRB protocol #20120821). Alle onderwerpen of hun wettelijke vertegenwoordigers ondertekenden de geïnformeerde toestemming. Commerci?...

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	<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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In deze studie werden twee in de handel verkrijgbare bibliotheek voorbereidings kits, voorbehandelings opties en input RNA-bedragen getest om een circRNA-verrijkings protocol voor de bouw van circRNA-sequencing-bibliotheken te optimaliseren. Op basis van de beoordelingen van deze studie, zijn een aantal belangrijke aspecten en kritieke stappen bij het maken van circRNA-sequencing bibliotheken duidelijk. Onze evaluatie bevestigt het nut van RNase R voor behandeling, zoals weergegeven door het toegenomen aantal Circrna's g...

Circulaire rna's (circrna's) zijn endogene, niet-Codeer rna's die aandacht hebben gekregen gezien hun pervasieve expressie in de eukaryote transcriptome1,2,3. Ze worden gevormd wanneer exonen terug-Splice aan elkaar en dus werden aanvankelijk beschouwd als splicing artefacten4,5. Recent onderzoek heeft echter aangetoond dat circrnas celtype, weefsel en ontwikkelingsfase specifieke expressie3,6 vertoont en evolutionair wordt bewaard op2,3. Bovendien zijn ze betrokken bij de bemiddeling van eiwit-eiwit interacties7, micro-RNA (Mirna) binding3,8,9,10, en regulering van ouderlijke genen transcriptie11.
In de klassieke RNA-sequencing (RNA-SEQ), kan circRNAs volledig verloren gaan tijdens de bibliotheek constructie als gevolg van poly-een selectie voor Mrna's of kan het moeilijk zijn om te isoleren gezien hun geringe overvloed. Recente circrna-karakterisering studies hebben echter een voorbehandelings stap opgenomen met behulp van RNase R om te verrijken voor circrnas2,12,13. RNase R is een exoribonuclease die lineaire Rna's verteerd, waardoor circulaire RNA-structuren achterblijven. CircRNA verrijking protocollen werden geoptimaliseerd door het genereren en vergelijken van gegevens van twee commercieel beschikbare hele transcriptome bibliotheek bouwkits, met en zonder een RNase R voor behandeling stap, en het gebruik van verschillende hoeveelheden totaal RNA input (1 tot 4 μg). Het geoptimaliseerde protocol werd vervolgens gebruikt om de overvloed aan Circrna's te evalueren in vijf verschillende hersengebieden (cerebellum [BC], inferieure Pariëtale kwab [IP], middelste temporele gyrus [MG], occipitale cortex [OC] en superieure frontale gyrus [SF]) en vier andere weefsel typen (lever [LV], Long [LU], lymfeklier [LN] en alvleesklier [PA]). RNA-SEQ-bibliotheken werden gekoppeld aan de eind geordend en de gegevens werden geanalyseerd met behulp van zes verschillende circrna Voorspellings algoritmen: find_circ3, ciri14, mapsplice15, Knife16, DCC17en circexplorer18. Op basis van onze analyse werd het hoogste aantal unieke Circrna's gedetecteerd bij het gebruik van een gestrande totale RNA Library Preparation Kit met RNase R voor behandeling en 4 μg totaal input RNA. Het geoptimaliseerde protocol wordt hier beschreven. Zoals eerder gemeld19,20, de hoogste verrijking van circrnas werd waargenomen in de hersenen in vergelijking met andere weefsel soorten.

<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

Circulaire Rna's (Circrna's) zijn een klasse van niet-Codeer Rna's die betrokken zijn bij functies zoals micro-RNA (miRNA)-regulering, bemiddeling van eiwit-eiwit interacties en regulering van de transcriptie van ouder genen. In de klassieke volgende generatie RNA sequencing (RNA-SEQ), Circrna's worden meestal over het hoofd gezien als gevolg van poly-een selectie tijdens de bouw van mRNA Bibliotheken, of zijn te vinden op zeer lage overvloed, en zijn daarom moeilijk te isoleren en op te sporen. Hier werd een circRNA Library Construction protocol geoptimaliseerd door het vergelijken van bibliotheek voorbereidings kits, pre-behandelingsopties en verschillende totaal RNA input bedragen. Twee in de handel verkrijgbare complete transcriptome bibliotheek voorbereidings kits, met en zonder RNase R voor behandeling, en het gebruik van variabele hoeveelheden RNA input (1 tot 4 μg), werden getest. Ten slotte, meerdere weefsel typen; waaronder lever, longen, lymfeklieren en pancreas; naast meerdere hersengebieden; met inbegrip van de cerebellum, inferieure Pariëtale kwab, middelste temporele gyrus, occipitale cortex, en superieure frontale gyrus; werden vergeleken om de overvloed van circRNA over weefsel typen te evalueren. Analyse van de gegenereerde RNA-SEQ-gegevens met behulp van zes verschillende circRNA-detectietools (find_circ, CIRI, Mapsplice, KNIFE, DCC en CIRCexplorer) onthulde dat een gestrande Total RNA Library-Voorbereidingspakket met RNase R voor behandeling en 4 μg RNA-input de optimale methode voor het identificeren van het hoogste relatieve aantal Circrna's. In overeenstemming met eerdere bevindingen werd de hoogste verrijking van circRNAs waargenomen in hersenweefsels in vergelijking met andere weefsel typen.

Gegevens die zijn gegenereerd met behulp van een in de handel verkrijgbare universele controle RNA (UC) en met behulp van twee bibliotheek voorbereidings kits, die beide een ribo-depletie-stap in hun protocollen bevatten, werden eerst beoordeeld. Met behulp van een analytische workflow (Data Analysis workflow, sectie 4), over het algemeen, werd een hoger aantal Circrna's gedetecteerd in de TruSeq datasets in vergelijking met de Kapa Ones (Figuur 1). Hoewel de ribosomale RNA (rRNA) percentage...

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

Identification of Circular RNAs using RNA Sequencing

Circulaire Rna's (Circrna's) zijn niet-Codeer Rna's die mogelijk rollen hebben in transcriptionele regulatie en die interacties tussen eiwitten bemiddel- Na beoordeling van verschillende parameters voor de bouw van circRNA sequencing Bibliotheken, een protocol werd gecompileerd met behulp van gestrande Total RNA Library voorbereiding met RNase R pre-behandeling en wordt hier gepresenteerd.

Identificatie van circulaire Rna's met RNA-sequencing

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

Dit protocol is het resultaat van de evaluatie van de belangrijkste aspecten van circRNA-bibliotheekvoorbereiding, zoals het type kit, RNase R-behandeling, invoerbedragen en hun impact op circRNA-detectie. Het maakt een optimale circRNA-detectie mogelijk en levert gegevens over aanpasbare parameters, afhankelijk van de behoeften van de gebruiker. Het identificeren van circRNAs in verschillende monstertypen zal ons helpen om de verdeling van hun expressie beter te begrijpen en stelt de weg in voor het afbakenen van de functionele rol van deze moleculen.Deze methode kan worden toegepast op elk totaal RNA-monster. Het is belangrijk om RNA-monsters op ijs te houden voordat het protocol wordt begin. Beoordeel de RIN- en DV200-waarden op de Agilent Bioanalyzer of TapeStation vóór de voorbereiding en volg de juiste procedures bij het werken met RNA.Begin met het verdunnen van het totale RNA tot vier microgram in 39 microliter RNase-vrij water en pipetten het op en neer te mengen. Gebruik in een aparte buis 1x RNase R Buffer om de RNase R te verdunnen tot een werkconcentratie van twee eenheden per microliter. Pipette 39 microliters van totale RNA en vijf microliters van 10x RNase R Reaction Buffer in een 1,5-microliter reactiebuis, en meng.Voeg zes microliter van de verdunde RNase R.Vervolgens de pipet aan te passen aan het volledige reactievolume en meng de oplossing door 10 keer op en neer te pipetten. Plaats de buis in een 37-graden Celsius waterbad gedurende 10 minuten, zorg ervoor dat het volledige reactievolume wordt ondergedompeld in het waterbad. Plaats vervolgens de buis op ijs en ga onmiddellijk verder met RNA-opruimen en concentratie.Bereid de RNA Wash Buffer voor voordat u begint met het mengen van het concentraat met 48 milliliter ethanol van 100% en plaats zuiveringskolommen in opvangbuizen. Voeg twee delen RNA Binding Buffer toe aan het RNase R-behandelde monster en meng goed. Voeg 150 microliter van 100% ethanol toe aan het mengsel voor een totaal van 300 microliter, breng het volledige volume over naar de kolom en vercentrifuge gedurende 30 seconden.Verwijder de stroom door, en voeg 400 microliter van RNA Prep Buffer rechtstreeks aan de kolom. Centrifuge gedurende 30 seconden, en gooi de stroom door. Voeg vervolgens 700 microliter RNA Wash Buffer toe aan de kolom, centrifuge gedurende 30 seconden en gooi de stroom door.Voeg nog eens 400 microliter van de Wasbuffer toe, centrifuge gedurende twee minuten en breng de kolom over naar een verse RNase-vrije buis van 1,5 milliliter. Voeg voorzichtig 11 microliter RNase-vrij water direct aan de kolom door de pipetpunt recht boven het kolomfilter te houden. Laat het monster een minuut op kamertemperatuur zitten en centrifuger het vervolgens gedurende één minuut.Zorg ervoor dat er stroom door in de 1,5-milliliter buis, en gooi de kolom. Het gezuiverde RNA-monster kan worden opgeslagen bij min 80 graden Celsius of onmiddellijk worden gebruikt voor de voorbereiding van de bibliotheek. Breng 10 microliter van het gezuiverde totale RNA over naar een schone put in een 96-well PCR-plaat.Voeg vijf microliters rRNA Binding Buffer toe, gevolgd door vijf microliters rRNA Removal Mix, enpipet vervolgens zachtjes op en neer om de reagentia te mengen. Verzegel de plaat en broed het vijf minuten uit bij 68 graden Celsius op een voorgeprogrammeerd en voorverwarmd thermocyclerblok. Laat de plaat na de incubatie een minuut op kamertemperatuur zitten.Verwijder de afdichting van de plaat en voeg 35 microliter vortexed kamertemperatuur rRNA verwijdering kralen aan het monster. Pas de pipet aan op 45 microliters en pipet 10 tot 20 keer op en neer om grondig te mengen. Laat de plaat een minuut op kamertemperatuur zitten.Breng de plaat naar een magnetische standaard en broed deze daar een minuut of totdat de oplossing is gewist. Breng de supernatant naar nieuwe goed op de plaat. Breng vervolgens de plaat van de magnetische standaard over naar de plaatstandaard.Vortex RNA opruimen kralen totdat ze goed zijn verspreid, en voeg 99 microliter kralen aan het monster. Pipette op en neer om te mengen, en de plaat in tecuniseren bij kamertemperatuur gedurende 10 minuten. Breng de plaat naar de magnetische standaard, en laat het daar voor vijf minuten of totdat de oplossing wist, verwijder en gooi alle supernatant uit de put.Met de plaat nog steeds op de magnetische standaard, voeg 200 microliters van vers bereide 80% ethanol aan de put zonder verstoring van de kralen. Laat de ethanol in de put gedurende 30 seconden, verwijder en gooi het. Voeg 11 microliter elution buffer aan de put, en pipette het op en neer te mengen.Broed de plaat twee minuten op kamertemperatuur, breng deze vervolgens terug naar de magnetische standaard en bewaar hem daar totdat de oplossing is opgelost. Breng 8,5 microliter van de supernatant naar een nieuwe put op dezelfde plaat, en voeg 8,5 microliter van Elute, Primer, Fragment High Mix aan elke put met een monster. Pipet op en neer 10 keer om grondig te mengen, verzegelen de plaat, en uitbroeden gedurende acht minuten in een thermocycler voorverwarmd tot 94 graden Celsius.Koel het monster tot vier graden Celsius, verwijder de plaat uit de thermocycler en centrifuge het kort. Twee bibliotheekvoorbereidingskits, TruSeq en KAPA, werden voor dit protocol beoordeeld. Over het algemeen werd een hoger aantal cirkel-RNAs en een lager percentage ribosomale RNA gedetecteerd bij het gebruik van de TruSeq-kit, dus TruSeq werd geselecteerd voor verdere experimenten.De betekenis van RNase R-voorbehandeling werd geëvalueerd door de gegevens te vergelijken die zijn gegenereerd uit voorbehandelde en niet-voorbehandelde bibliotheken. Zoals verwacht werd in de voorbehandelde bibliotheken consequent een groter aantal circulaire RNA's geïdentificeerd in vergelijking met niet-voorbehandelde bibliotheken. De hoeveelheid input RNA werd geoptimaliseerd voor het detecteren van een hogere diversiteit aan circulaire RNAs.Bibliotheken werden bereid met behulp van een, twee en vier microgram van input RNA, en de hoogste diversiteit van circulaire RNA soorten werd waargenomen bij het gebruik van vier microgram van het totale RNA. Het geoptimaliseerde protocol werd gebruikt om circulaire RNA overvloeden te vergelijken in vijf hersengebieden van vier gezonde donoren en voor andere weefselsoorten van zes gezonde donoren. Over het algemeen werd een hogere overvloed aan circulaire RNase waargenomen in de hersenen in vergelijking met andere weefseltypen.Het is belangrijk om te onthouden om de juiste procedures te volgen bij het werken met RNA, met inbegrip van het dragen van handschoenen, het grondig reinigen van de bank en pipetten met RNase doekjes of spray voordat u het protocol, en het houden van RNA op ijs vooraf. Na deze procedure kan orthogonale validatie worden uitgevoerd, zoals Sanger sequencing van geïdentificeerde circRNA-knooppunten. De ontdekking van nieuwe circRNAs en verder bewijs van hun aanwezigheid kerft een nieuw onderzoeksgebied voor het verkennen van hun biologische functies.

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.

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.

Metabole Labeling en profilering van overdracht RNAs met behulp van 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.

Retrospectieve MicroRNA Sequencing: CDNA Bibliotheek voorbereiding Protocol met behulp van formaline-vaste paraffine-ingebedde RNA Specimens

&#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.

Zeldzame gebeurtenis detectie met behulp van fout-gecorrigeerd DNA en RNA Sequencing

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.

High-throughput identificatie van regelgevende gensequenties met behulp van Next-generation Sequencing van circulaire chromosoom conformatie 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 ...

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 met behulp van gen-activeren 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.

Het rangschikken van mRNA van geheel bloed gebruikend Nanogaatje het rangschikken

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.

Eencellige RNA-sequencing en analyse van menselijke pancreatic eilandjes

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).

Identificatie van voetafdrukken van RNA: eiwitcomplexen via RNA-immunoprecipitatie in tandem gevolgd door 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 ...

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.

Verbetering van kleine RNA-SEQ: minder bias en betere detectie van 2 '-O-methyl Rna's

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 ...