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 are grateful to the Banner Sun Health Research Institute Brain and Body Donation Program (BBDP) of Sun City, Arizona for the provision of human brain tissues. The BBDP has been supported by the National Institute of Neurological Disorders and Stroke (U24 NS072026 National Brain and Tissue Resource for Parkinson's Disease and Related Disorders), the National Institute on Aging (P30AG19610 Arizona Alzheimer's Disease Core Center), the Arizona Department of Health Services (contract 211002, Arizona Alzheimer's Research Center), the Arizona Biomedical Research Commission (contracts 4001, 0011, 05-901 and 1001 to the Arizona Parkinson&#39;s Disease Consortium) and the Michael J. Fox Foundation for Parkinson's Research27. This study was also supported by the DHS and the State of Arizona (ADHS grant # ADHS14-052688). We also thank Andrea Schmitt (Banner Research) and Cynthia Lechuga (TGen) for administrative support.

This research has been performed in compliance with all institutional, national and international guidelines for human welfare. Brain tissues were obtained from the Banner Sun Health Research Institute Brain and Body Donation Program in Sun City, AZ. The operations of the Brain and Body Donation Program are approved by the Western Institutional Review Board (WIRB protocol #20120821). All subjects or their legal representatives signed the informed consent. Commercial (non-brain) biospecimens were purchased from Proteogene...

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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 this study, two commercially available library preparation kits, pre-treatment options, and input RNA amounts were tested in order to optimize a circRNA enrichment protocol for construction of circRNA sequencing libraries. Based on this study&rsquo;s assessments, a number of key aspects and critical steps in creating circRNA sequencing libraries are apparent. Our evaluation confirms the utility of RNase R pre-treatment, as reflected by the increased number of circRNAs detected. Overall, a higher diversity of circRNAs ...

Circular RNAs (CircRNAs) are endogenous, non-coding RNAs that have gained attention given their pervasive expression in the eukaryotic transcriptome1,2,3. They are formed when exons back-splice to each other and hence were initially considered to be splicing artifacts4,5. However, recent studies have demonstrated that circRNAs exhibit cell type, tissue, and developmental stage specific expression3,6 and are evolutionarily conserved2,3. Furthermore, they are involved in mediation of protein-protein interactions7, micro-RNA (miRNA) binding3,8,9,10, and regulation of parental gene transcription11.
In classical RNA sequencing (RNA-seq), circRNAs may be completely lost during library construction as a result of poly-A selection for mRNAs or may be difficult to isolate given their low abundance. However, recent circRNA characterization studies have incorporated a pre-treatment step using RNase R in order to enrich for circRNAs2,12,13. RNase R is an exoribonuclease that digests linear RNAs, leaving behind circular RNA structures. CircRNA enrichment protocols were optimized by generating and comparing data from two commercially available whole transcriptome library construction kits, with and without an RNase R pre-treatment step, and using varying amounts of total RNA input (1 to 4 &#181;g). The optimized protocol was next used to evaluate the abundance of circRNAs across five different brain regions (cerebellum [BC], inferior parietal lobe [IP], middle temporal gyrus [MG], occipital cortex [OC] and superior frontal gyrus [SF]) and four other tissue types (liver [LV], lung [LU], lymph node [LN] and pancreas [PA]). RNA-seq libraries were paired end sequenced and data was analyzed using six different circRNA prediction algorithms: find_circ3, CIRI14, Mapsplice15, KNIFE16, DCC17, and CIRCexplorer18. Based on our analysis, the highest number of unique circRNAs was detected when using a stranded total RNA library preparation kit with RNase R pre-treatment and 4 &#181;g total input RNA. The optimized protocol is described here. As previously reported19,20, the highest enrichment of circRNAs was observed in the brain compared to other tissue types.

<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) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein interactions, and regulation of parental gene transcription. In classical next generation RNA sequencing (RNA-seq), circRNAs are typically overlooked as a result of poly-A selection during construction of mRNA libraries, or are found at very low abundance, and are therefore difficult to isolate and detect. Here, a circRNA library construction protocol was optimized by comparing library preparation kits, pre-treatment options and various total RNA input amounts. Two commercially available whole transcriptome library preparation kits, with and without RNase R pre-treatment, and using variable amounts of total RNA input (1 to 4 &#181;g), were tested. Lastly, multiple tissue types; including liver, lung, lymph node, and pancreas; as well as multiple brain regions; including the cerebellum, inferior parietal lobe, middle temporal gyrus, occipital cortex, and superior frontal gyrus; were compared to evaluate circRNA abundance across tissue types. Analysis of the generated RNA-seq data using six different circRNA detection tools (find_circ, CIRI, Mapsplice, KNIFE, DCC, and CIRCexplorer) revealed that a stranded total RNA library preparation kit with RNase R pre-treatment and 4 &#181;g RNA input is the optimal method for identifying the highest relative number of circRNAs. Consistent with previous findings, the highest enrichment of circRNAs was observed in brain tissues compared to other tissue types.

Data generated using a commercially available universal control RNA (UC) and using two library preparation kits, both of which include a ribo-depletion step in their protocols, was first assessed. Using an analytical workflow (Data analysis workflow, section 4), overall, a higher number of circRNAs was detected in the TruSeq datasets compared to the Kapa ones (Figure 1). Although the ribosomal RNA (rRNA) percentages were below 5% in datasets from both kits for lower input amounts (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

Circular RNAs (circRNAs) are non-coding RNAs that may have roles in transcriptional regulation and mediating interactions between proteins. Following assessment of different parameters for construction of circRNA sequencing libraries, a protocol was compiled utilizing stranded total RNA library preparation with RNase R pre-treatment and is presented here.

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

This protocol is the result of evaluating key aspects of circRNA library preparation, such as kit type, RNase R treatment, input amounts and their impact on circRNA detection. It enables optimal circRNA detection and provides data on adjustable parameters depending on the needs of the user. Identifying circRNAs in different sample types will help us to better understand the distribution of their expression and sets the stage for delineating the functional role of these molecules.This method can be applied to any total RNA sample. It is important to keep RNA samples on ice prior to beginning the protocol. Assess the RIN and DV200 values on the Agilent Bioanalyzer or TapeStation before the prep, and follow appropriate procedures when working with RNA.Begin by diluting total RNA to four micrograms in 39 microliters of RNase-free water and pipetting it up and down to mix. In a separate tube, use 1x RNase R Buffer to dilute the RNase R to a working concentration of two units per microliter. Pipette 39 microliters of total RNA and five microliters of 10x RNase R Reaction Buffer into a 1.5-microliter reaction tube, and mix.Add six microliters of the diluted RNase R.Then adjust the pipette to full reaction volume, and mix the solution by pipetting up and down 10 times. Place the tube in a 37-degree Celsius water bath for 10 minutes, making sure that the full reaction volume is immersed in the water bath. Then, place the tube on ice, and immediately proceed with RNA cleanup and concentration.Prior to starting, prepare the RNA Wash Buffer by mixing the concentrate with 48 milliliters of 100%ethanol, and place purification columns into collection tubes. When ready, add two volumes of RNA Binding Buffer to the RNase R-treated sample, and mix well. Add 150 microliters of 100%ethanol to the mixture for a total of 300 microliters, transfer the entire volume to the column, and centrifuge for 30 seconds.Remove the flow through, and add 400 microliters of RNA Prep Buffer directly to the column. Centrifuge for 30 seconds, and discard the flow through. Then, add 700 microliters of RNA Wash Buffer to the column, centrifuge for 30 seconds, and discard the flow through.Add another 400 microliters of the Wash Buffer, centrifuge for two minutes, and transfer the column to a fresh RNase-free 1.5-milliliter tube. Carefully add 11 microliters of RNase-free water directly to the column by holding the pipette tip right above the column filter. Let the sample sit for one minute at room temperature, and then centrifuge it for one minute.Make sure there is flow through in the 1.5-milliliter tube, and discard the column. The purified RNA sample can be stored at minus 80 degrees Celsius or used immediately for library preparation. Transfer 10 microliters of the purified total RNA to a clean well in a 96-well PCR plate.Add five microliters of rRNA Binding Buffer followed by five microliters of rRNA Removal Mix, then gently pipette up and down to mix the reagents. Seal the plate, and incubate it for five minutes at 68 degrees Celsius on a preprogrammed and preheated thermocycler block. After the incubation, let the plate sit at room temperature for one minute.Remove the seal from the plate, and add 35 microliters of vortexed room temperature rRNA removal beads to the sample. Adjust the pipette to 45 microliters, and pipette up and down 10 to 20 times to thoroughly mix. Let the plate sit at room temperature for one minute.Transfer the plate to a magnetic stand, and incubate it there for one minute or until the solution clears. Transfer the supernatant to new well on the plate. Then transfer the plate from the magnetic stand to the plate stand.Vortex RNA cleanup beads until they are well dispersed, and add 99 microliters of beads to the sample. Pipette up and down to mix, and incubate the plate at room temperature for 10 minutes. Transfer the plate to the magnetic stand, and leave it there for five minutes or until the solution clears, then remove and discard all supernatant from the well.With the plate still on the magnetic stand, add 200 microliters of freshly prepared 80%ethanol to the well without disrupting the beads. Leave the ethanol in the well for 30 seconds, then remove and discard it. Add 11 microliters of Elution Buffer to the well, and pipette it up and down to mix.Incubate the plate at room temperature for two minutes, then transfer it back to the magnetic stand, and keep it there until the solution clears. Transfer 8.5 microliters of the supernatant to a new well on the same plate, and add 8.5 microliters of Elute, Primer, Fragment High Mix to each well containing a sample. Pipette up and down 10 times to mix thoroughly, seal the plate, and incubate it for eight minutes in a thermocycler preheated to 94 degrees Celsius.Cool the sample to four degrees Celsius, remove the plate from the thermocycler, and centrifuge it briefly. Two library preparation kits, TruSeq and KAPA, were assessed for this protocol. Overall, a higher number of circular RNAs and lower percentage of ribosomal RNA was detected when using the TruSeq kit, so TruSeq was selected for further experiments.The significance of RNase R pretreatment was evaluated by comparing the data generated from pretreated and non-pretreated libraries. As expected, a higher number of circular RNAs was consistently identified in the pretreated libraries compared to non-pretreated ones. The amount of input RNA was optimized for detecting a higher diversity of circular RNAs.Libraries were prepared using one, two, and four micrograms of input RNA, and the highest diversity of circular RNA species was observed when using four micrograms of total RNA. The optimized protocol was used to compare circular RNA abundances in five brain regions from four healthy donors and for other tissue types from six healthy donors. Overall, a higher abundance of circular RNase was observed in the brain compared to other tissue types.It is important to remember to follow appropriate procedures when working with RNA, including wearing gloves, thoroughly cleaning the bench and pipettes with RNase wipes or spray before beginning the protocol, and keeping RNA on ice beforehand. Following this procedure, orthogonal validation such as Sanger sequencing of identified circRNA junctions can be performed. The discovery of new circRNAs and further evidence of their presence carves out a new area of research for exploring their biological functions.

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 ...

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 ...

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 ...

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 ...

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

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 ...

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 ...

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, ...

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 ...

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

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 ...

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 ...