This work was supported by the Department of Defense DoD grant AZ180075. Stefan Stamm thanks Jacqueline Noonan Endowment. Anna Pawluchin was supported by the DAAD, German academic exchange program, Justin R. Welden was a recipient of the University of Kentucky Max Steckler Award.

1. Design of the constructs
<ol>
	<li>Use the UCSC genome browser24 to identify repetitive elements necessary for circular RNA formation and incorporate them in the constructs. Importantly, primers for amplification need to be outside the repetitive elements.</li>
	<li>Paste the circular RNA sequence (Supplemental Figure 1 is a test sequence) into <a href="https://genome.ucsc.edu/cgi-bin/hgBlat?command=start" target="_blank">https://genome.ucsc.edu/cgi-bin/hgBlat?command=start</a> and select the right organism. Submit the sequence and go to browser view, zoom out 1.5x or as appropriate (Figure 2A). 
	NOTE: The search sequence appears in the top line (Figure 2A, 1). Depending on the order of the exons in the circular RNA, BLAT will not connect all the exons. In this example, exon 12 (Figure 2A, 4) is not connected to 11 (Figure 2A, 2, 3), because exon 12 is upstream of exon 11 in the circular RNA sequence (Supplemental Figure 1). The repetitive elements are in the &#39;repeat masker&#39; track, indicated by boxes, where black to gray color indicates the evolutionary conservation (Figure 2A, 5).</li>
	<li>Mouse over the repetitive elements to identify their subtype in a floating window. Alu elements are in the SINE (short interspersed nuclear element) line. Use the &#39;default tracks&#39; button under the window to reset the browser if a different picture than Figure 2 is obtained. 
	NOTE: Mousing over the exons in the gene display generates a window with exon numbers that are computer generated. These numbers do not correspond to the exon numbering established in the literature and also change between isoforms.</li>
</ol>
2. Select the sequence to be cloned in an expression vector
<ol>
	<li>Download the DNA sequence shown in the window by going to View &#8594; DNA on the top line of the UCSC genome browser. In the Sequencing Formatting Option, select Extended Case/Color Options.</li>
	<li>Select the default case as Lower and select toggle case for NCBI refseq. Select underline, and bold, and italic for Repeat Masker. Click Submit. There will be exons as capital letters and introns as small letters. Check the exon/intron borders.</li>
	<li>In this example there is a &#39;ccctttacCTTTTT&#39; sequence, indicating that the browser shows the reverse complement. If this is the case, go back and select the reverse complement box until seeing the correct exon intron borders (agEXONgt), in this example AAAAAGgtaaaggg.</li>
	<li>Copy the file with the correct orientation (internal exons are surrounded by intronic ag&#8230;gt) into a word processing document and highlight the exons (Supplemental Figure 2). 
	NOTE: In this example, the genomic fragment encompassing exons 9-12 is around 24 kb and thus too large to be amplified from genomic DNA. Therefore, each exon surrounded by about 1 kb intronic region is individually amplified and these four fragments are assembled in a cloning vector.</li>
	<li>Select fragments to be amplified (exon &#177; 500 nt intron). Make sure that the intron does not begin or end in a repetitive region, as primers in these regions will not amplify specific sequences. The selected regions are shown in Figure 2B, and their sequences are shown in Supplemental Figure 3. 
	NOTE: In general, the larger the constructs, the more difficult the cloning will be. The fragments can be assembled either step wise (i.e., exon 9 is combined with the vector and in the next step exon 10 is introduced into this construct via cloning until all exons are in place), or alternatively, all fragments are assembled simultaneously. A stepwise approach always works but requires more time. A simultaneous assembly does not always work and depends on how well the individual fragments can be amplified from genomic DNA and on the overall size of the construct. We therefore usually start with both approaches simultaneously.</li>
</ol>
3. Design primers for cloning
<ol>
	<li>Use a web tool (<a href="https://nebuilder.neb.com/#!/" target="_blank">https://nebuilder.neb.com/#!/</a>) to design the primers for cloning. For example, enter fragments 9, 10, 11 and 12 and the vector sequence (Supplemental Figure 3) to this tool.</li>
	<li>For the vector sequence, add the insertion site as the last nucleotide and subsequently add the fragments. Since the vector numbering does not start with a given insertion site, the site of insertion in the vector is located and the downstream part is put in in front of the upstream sequence. In this example the inserts start directly after the HindIII [AAGCTT] site and ends directly after the PmeI [GTTTAAAC] site of pcDNA3.1. The sequence from cccgctgatcag &#8230; ccgtaaaaaggccgc is pasted in front of position 1 in the pcDNA3.1 sequence (gttgctggcgtttttcc &#8230;).</li>
	<li>Adjust primers if their melting points are more than 4 &#176;C apart, as they will not work in amplification. The assembly of the fragments and primer sequences designed are shown in Supplemental Figure 4. Primers for cloning can also be designed manually25.</li>
</ol>
4. PCR and amplicon detection
<ol>
	<li>Standard PCR Reaction: Make a reaction mix for total volume of 50 &#181;L per reaction. Below is the recipe for one reaction using polymerase 1: 
	10 &#181;L of 5x Reaction Buffer 
	1 &#181;L of 10 mM dNTPs 
	2.5 &#181;L of 10 &#181;M Forward Primer 
	2.5 &#181;L of 10 &#181;M Reverse Primer 
	0.5 &#181;L of Polymerase 1 
	32.5 &#181;L of Nuclease free H2O
	<ol>
		<li>Optionally, add 10 &#181;L of 5x GC Enhancer if the product has high GC content; make a separate mix containing GC Enhancer to test PCR reaction.</li>
	</ol>
	</li>
	<li>Aliquot 49 &#181;L of the mix into PCR tubes per reaction sample.</li>
	<li>Add 1 &#181;L of DNA to the PCR, the amount ranges from 10 pg to 1 ng.</li>
	<li>Spin down the samples to remove residue off the sides and place them in a PCR machine. Use the same machine as well as same spots in the machine when optimizing the PCR conditions.</li>
</ol>
5. Optimization for longer DNA fragments for use of different polymerases
<ol>
	<li>Temperature
	<ol>
		<li>Optimize the long-range Polymerase 2.
		<ol>
			<li>Use a lower denaturation temperature (Polymerase 1: 98 &#176;C, Polymerase 2: 94 &#176;C).</li>
			<li>Use a longer denaturation time (Polymerase 1: 10 s, Polymerase 2: 30 s).</li>
			<li>Use a longer annealing time (Polymerase 1: 30 s, Polymerase 2: 60 s).</li>
			<li>Use a longer extension time (Polymerase 1: 30 s/kb, Polymerase 2: 50 s/kb).</li>
			<li>Use a lower extension temperature (Polymerase 1: 72 &#176;C, Polymerase 2: 65 &#176;C).</li>
			<li>Use an annealing temperature 5 &#176;C below the Tm of the primers.</li>
		</ol>
		</li>
		<li>Optimize the primer concentrations (5x less to 5x more than the original primer concentration). Optimize DNA concentrations from 10 pg to 50 pg, 100 pg, 1 ng, 5 ng, 10 ng.</li>
		<li>As an example of a PCR program to amplify a 15 kb product size using Polymerase 2, perform an initial denaturation at 94 &#176;C for 30 s, DNA denaturation at 94 &#176;C for 30 s followed by annealing at 58 &#176;C for 30 s, and DNA extension at 65 &#176;C for 12 min 30 s. Perform a final extension at 65 &#176;C for 10 min with a final 4 &#176;C hold. 
		NOTE: The annealing temperature (Ta) specific for the primers determined by web-based temperature calculations that are specific for each polymerase. Extension times may vary and need to be optimized if fragments are larger than 10 kb.</li>
	</ol>
	</li>
	<li>Extension times and DNA concentrations
	<ol>
		<li>Use longer extension times for DNA fragments over 6 kb: 1 min per 1 kb. Longer fragments should use less DNA.</li>
		<li>Perform dilutions of DNA to find the optimum DNA concentration for amplification, usually 1 pg to 1 ng for plasmids or 1 ng to 1 &#181;g for genomic DNA. 
		NOTE: For most cloning use polymerase 1, engineered by fusing the Sso7d DNA binding domain to a proprietary thermostable DNA polymerase26. The polymerase has a low error rate and due to the Sso7d domain a high processivity, needed to amplify large (10-15 kb) genomic fragments. Due to this large fragment size, enzymatic assembly of DNA molecules27 is used for insertion into vectors, as this method does not require restriction enzymes. The amplification of fragments longer than 15 kb gets increasingly difficult with polymerase 1. For large fragment amplification use polymerase 2 made from pyrococcus-like proofreading polymerase fused to Sso7d or the long range PCR kit that gives, however, higher error rates.</li>
	</ol>
	</li>
</ol>
6. Purification of PCR products for cloning
<ol>
	<li>Run half of the PCR products on 1% agarose gels containing an intercalating nucleic acid stain (e.g., 1x GelGreen). Visualize the stained gels on a dark reader transilluminator. This stained DNA does not run true to size. 
	NOTE: GelGreen intercalates into DNA, similar to ethidium bromide, but it is excited by light around 500 nm (cyan), a wavelength that does not damage DNA, which highly improves cloning.</li>
	<li>In parallel, run half of the PCR products on a 1% gel that is stained post-run with ethidium bromide (Figure 3A,B).</li>
	<li>Excise bands of the right size from the stained gel (step 6.1) and isolate DNA using a gel and PCR cleanup kit. In deviation from its standard protocol, elute the DNA in only 20 &#181;L of double distilled water, as usually the DNA concentrations are low.</li>
	<li>Prior to cloning, check the isolated DNA on an agarose gel for concentration and integrity (Figure 3B). Aim for a 2:1 molar insert to vector ratio. When using several inserts, the ratio is 2:2:2:1.</li>
</ol>
7. DNA assembly and clone detection
NOTE: Cloning is done using an enzymatic DNA assembly kit, with minor modifications. The assembly is performed for 60 min at 50 &#176;C and generally the lower range of DNA is used for assembly (20-100 fmol/20 &#181;L reaction). The whole reaction mix is added to chemical competent cells and the whole cells plated out on a 6 cm agar plate.
<ol>
	<li>Combine the vector and insert in a 1:2 molar ratio (20-500 fmol each) in 10 &#181;L of water.</li>
	<li>Add 10 &#181;L of DNA Assembly Master Mix.</li>
	<li>Incubate samples for 60 minutes at 50 &#176;C.</li>
	<li>Next, transform competent cells with the total assembly reaction. Here, use E. coli strain 1 cells for shorter constructs or E. coli strain 2 cells for longer or unstable constructs. Cells should be in a 50 &#181;L volume.</li>
	<li>Thaw cells on ice and add 2 &#181;L of the chilled assembled product to the competent cells. Mix by gently flicking the tube 4-5 times. Do not vortex.</li>
	<li>Let the mixture sit on ice for 30 min.</li>
	<li>Heat shock at 42 &#176;C for 30 s. Do not mix.</li>
	<li>Transfer the tube back to ice for 2 min.</li>
	<li>Add 950 &#181;L of room-temperature SOC Media to the tube.</li>
	<li>Incubate the reaction tube at 37 &#176;C for 60 min. Shake vigorously (300 rpm).</li>
	<li>Warm selection plates with the appropriate antibiotic during incubation at 37 &#176;C.</li>
	<li>Pellet the cells through centrifugation (10,000 x g, 30 s) and plate out &#188; and &#190; of cells on two selection plates and incubate at 37 &#176;C overnight.</li>
</ol>
8. Validation of clones
<ol>
	<li>Use colony PCR, employing PCR primers spanning the assembly sites (Figure 1C) for detection.</li>
	<li>Take a sterile toothpick and touch a single bacterial colony.</li>
	<li>Touch the bottom of a PCR tube with the toothpick that has the colony and then streak the toothpick on an antibiotic agar plate, incubate the streaked plate at 37 &#176;C or room temperature for sensitive constructs overnight.</li>
	<li>Overlay the PCR tube touched with the colony with a PCR mix containing the detection primers. These are designed using primer 3 (<a href="http://bioinfo.ut.ee/primer3-0.4.0/" target="_blank">http://bioinfo.ut.ee/primer3-0.4.0/</a>). The program has the option to force primer design across a defined sequence using the &#34;[ccc]&#34; signs to select primers across the assembly junction. DNA from positive strains is isolated and verified by sequencing.</li>
</ol>
9. Analysis of circular RNA expressing reporter genes
<ol>
	<li>For analysis, transfect reporter genes into eukaryotic cells. Here, use HEK293 cells (ATTC #CRL-1573) as they give high transfection efficiency. To save costs, use PEI (polyethyleneimine) solutions.</li>
	<li>For the PEI solution, dissolve linear polyethyleneimine hydrochloride (PEI) at 1 mg/mL in water at low pH, pH 2. Bring up the pH to 7 with NaOH. Sterile filter with 0.22 &#181;m filters and store at 4 &#176;C.</li>
	<li>Split cells into six wells (approximately 150,000 cells per well) and let them grow overnight in 10% FBS in DMEM media.</li>
	<li>Aliquot 1 &#181;g of the reporter gene in a sterile tube and add 200 &#181;L of sterile filtered 150 mM NaCl. Mix with the DNA by vortexing.</li>
	<li>Add PEI solution to this mix and vortex, briefly centrifuge to collect samples at bottom of tube. Use a ratio of 1 &#181;g of DNA per 3 &#181;L of PEI.</li>
	<li>Incubate at room temperature for 10 min and then add directly to HEK 293 cells.</li>
	<li>Incubate HEK293 cells at 37 &#176;C, 5% CO2, overnight.</li>
	<li>Isolate RNA for RT-PCR via an RNA isolation kit.</li>
</ol>
10. RNase R treatment to remove linear RNAs
<ol>
	<li>Use 10 &#181;g of total RNA in an RNase-free tube.</li>
	<li>Add 10 &#181;L of 10x RNase R buffer (0.2 M Tris-HCl (pH 8.0), 1 M KCl, 1 mM MgCl2) to RNA.</li>
	<li>Add RNase R to RNA (1 &#181;l = 20U).</li>
	<li>Add 1 &#181;L of glycol blue to the RNA and bring the volume up to 100 &#181;L with sterile water.</li>
	<li>Incubate samples at 37 &#176;C for 30 min.</li>
	<li>Add 100 &#181;L of phenol/chloroform and vortex for 1 min.</li>
	<li>Centrifuge at 21,000 x g for 1 min to separate phases.</li>
	<li>Take the supernatant (aqueous phase) and add 1 volume (around 80 &#181;L of chloroform).</li>
	<li>Vortex for 1 min, and then centrifuge for 1 min to separate phases.</li>
	<li>Take supernatant, add 1:10 vol KAc and 2.5 vol ethanol, and precipitate at -20 &#176;C for 1-4 h. Centrifuge at 4 &#176;C for 30 min at full speed (21,000 x g). There will be a small blue pellet at the bottom.</li>
	<li>Remove the supernatant, and wash with 80% ethanol. Let air dry for 5 min at room temperature, and dissolve in 10 &#181;L water.</li>
</ol>
11. RT-PCR analysis
<ol>
	<li>Use 1 &#181;g of RNA per RT reaction.</li>
	<li>Make reaction mix for a final total volume of 20 &#181;L per reaction. For one reaction, use 1 &#181;L of 10 mM dNTPs, 1 &#181;L of 0.1 M Dithiothreitol (DTT), 4 &#181;L of 5x First-Strand Buffer, and 0.5 &#181;L of Reverse Transcriptase.</li>
	<li>Aliquot 6.5 &#181;L of reaction mix into new PCR tubes.</li>
	<li>Mix up to 5 primers in one mix. However, some primers may mis-pair with other primers in the PCR (Figure 5). Primer sequences are in Table 2. We routinely use gene-specific exon-junction primers but priming with random hexamers is also possible.</li>
	<li>Add 1 &#181;L of 10 &#181;M Reverse primer to desired RT reaction tube.</li>
	<li>Add 1 &#181;g of RNA to PCR reaction tube.</li>
	<li>Add RNase-free H2O up to a total volume of 20 &#181;L.</li>
	<li>Spin tubes down to remove residue on side of tubes and place in thermocycler.</li>
	<li>Run the RT reaction in thermocycler at 50 &#176;C for 50 minutes.</li>
	<li>Store RT cDNA at -20 &#176;C or proceed to the PCR reaction.</li>
</ol>

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nature Methods. 6 (5), 343-345 (2009).</li><li>Conn, S. J., 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=The+RNA+binding+protein+quaking+regulates+formation+of+circRNAs.">The RNA binding protein quaking regulates formation of circRNAs.</a> Cell. 160 (6), 1125-1134 (2015).</li><li>Jeck, W. R., Sharpless, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+and+characterizing+circular+RNAs.">Detecting and characterizing circular RNAs.</a> Nature Biotechnology. 32 (5), 453-461 (2014).</li><li>Pamudurti, N. 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=Translation+of+CircRNAs.">Translation of CircRNAs.</a> Molecular Cell. 66 (1), 9-21 (2017).</li><li>Kramer, M. C., 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=Combinatorial+control+of+Drosophila+circular+RNA+expression+by+intronic+repeats,+hnRNPs,+and+SR+proteins.">Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins.</a> Genes &amp; Development. 29 (20), 2168-2182 (2015).</li><li>Starke, 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=Exon+circularization+requires+canonical+splice+signals.">Exon circularization requires canonical splice signals.</a> Cell Reports. 10 (1), 103-111 (2015).</li><li>Suzuki, H., Tsukahara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+view+of+pre-mRNA+splicing+from+RNase+R+resistant+RNAs.">A view of pre-mRNA splicing from RNase R resistant RNAs.</a> International Journal of Molecular Sciences. 15 (6), 9331-9342 (2014).</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=Diverse+alternative+back-splicing+and+alternative+splicing+landscape+of+circular+RNAs.">Diverse alternative back-splicing and alternative splicing landscape of circular RNAs.</a> Genome Research. 26 (9), 1277-1287 (2016).</li><li>Liang, D., Wilusz, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+intronic+repeat+sequences+facilitate+circular+RNA+production.">Short intronic repeat sequences facilitate circular RNA production.</a> Genes &amp; Development. 28 (20), 2233-2247 (2014).</li><li>Wang, Y., Mandelkow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+in+physiology+and+pathology.">Tau in physiology and pathology.</a> Nature Reviews Neuroscience. 17 (1), 5-21 (2016).</li><li>Fejes-Toth, 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=Post-transcriptional+processing+generates+a+diversity+of+5'-modified+long+and+short+RNAs.">Post-transcriptional processing generates a diversity of 5'-modified long and short RNAs.</a> Nature. 457 (7232), 1028-1032 (2009).</li><li>Gao, Q. 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=Complex+regulation+of+tau+exon+10,+whose+missplicing+causes+frontotemporal+dementia.">Complex regulation of tau exon 10, whose missplicing causes frontotemporal dementia.</a> Journal of Neurochemistry. 74 (2), 490-500 (2000).</li><li>Hartmann, A. 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=Regulation+of+alternative+splicing+of+human+tau+exon+10+by+phosphorylation+of+splicing+factors.">Regulation of alternative splicing of human tau exon 10 by phosphorylation of splicing factors.</a> Molecular and Cellular Neuroscience. 18 (1), 80-90 (2001).</li><li>Wang, Y., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+backsplicing+produces+translatable+circular+mRNAs.">Efficient backsplicing produces translatable circular mRNAs.</a> RNA. 21 (2), 172-179 (2015).</li><li>Yang, Y., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constructing+GFP-Based+Reporter+to+Study+Back+Splicing+and+Translation+of+Circular+RNA.">Constructing GFP-Based Reporter to Study Back Splicing and Translation of Circular RNA.</a> Methods in Molecular Biology. 1724, 107-118 (2018).</li><li>Zheng, Q., 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+RNA+profiling+reveals+an+abundant+circHIPK3+that+regulates+cell+growth+by+sponging+multiple+miRNAs.">Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs.</a> Nature Communications. 7, 11215 (2016).</li><li>Jia, W., Xu, B., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNA+expression+profiles+of+mouse+ovaries+during+postnatal+development+and+the+function+of+circular+RNA+epidermal+growth+factor+receptor+in+granulosa+cells.">Circular RNA expression profiles of mouse ovaries during postnatal development and the function of circular RNA epidermal growth factor receptor in granulosa cells.</a> Metabolism. 85, 192-204 (2018).</li><li>Liang, D., 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=The+Output+of+Protein-Coding+Genes+Shifts+to+Circular+RNAs+When+the+Pre-mRNA+Processing+Machinery+Is+Limiting.">The Output of Protein-Coding Genes Shifts to Circular RNAs When the Pre-mRNA Processing Machinery Is Limiting.</a> Molecular Cell. 68 (5), 940-954 (2017).</li><li>Legnini, I., 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=Circ-ZNF609+Is+a+Circular+RNA+that+Can+Be+Translated+and+Functions+in+Myogenesis.">Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis.</a> Molecular Cell. 66 (1), 22-37 (2017).</li><li>Li, X., 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=Coordinated+circRNA+Biogenesis+and+Function+with+NF90/NF110+in+Viral+Infection.">Coordinated circRNA Biogenesis and Function with NF90/NF110 in Viral Infection.</a> Molecular Cell. 67 (2), 214-227 (2017).</li><li>Zhang, Y., 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=The+Biogenesis+of+Nascent+Circular+RNAs.">The Biogenesis of Nascent Circular RNAs.</a> Cell Reports. 15 (3), 611-624 (2016).</li></ol>

Nothing to disclose.

In general, circular RNAs are low abundant1, which complicates the study of their function and formation. Similar to linear RNAs13, the use of reporter minigenes allows the identification of cis and trans-acting factors that regulate the formation of circular RNAs. Thus, this approach generates hypotheses that can be further tested using the endogenous genes.
The most critical step is the design of the reporter gene. The enzymatic assembly of DNA...

Circular RNAs 
Circular RNAs (circRNAs) are covalently closed single stranded RNAs that are expressed in most organisms. They are generated by joining a downstream 5&#39; splice site to an upstream 3&#39; splice site, a process called back-splicing (Figure 1A)1. Sequences in the pre-mRNA that exhibit base complementary as short as 30-40 nt bring back-splice sites into proper alignment for circRNA formation2. In humans, Alu elements1, representing about 11% of the genome3, form extensive double stranded RNA structures in pre-mRNA due to their self-complementarity4,5 and thus promote the formation of circRNAs1.
Currently, three major functions of circRNAs have been described. Some circRNAs bind microRNAs (miRNAs) and through sequestration act like miRNA sponges6. CircRNAs have been implicated in transcriptional and post transcriptional regulation, through competition with linear splicing7 or modulation of transcription factor activity8. Finally, circRNAs contain short open reading frames and proof of principle studies show that they can be translated9,10. However, the function of most circRNAs remains enigmatic. The majority of circular RNAs have been detected using next-generation sequencing methods11. Detailed analyses of individual genes using targeted RT-PCR approaches reveal that a large number of circular RNAs remains to be discovered12.
Use of reporter genes to analyze pre-mRNA processing 
The analysis of mRNA derived from DNA reporter constructs transfected into cells is a well-established method to study alternative pre-mRNA splicing, which can be applied to circular RNAs. In general, the alternative exon, its surrounding introns, and constitutive exons are amplified and cloned into a eukaryotic expression vector. Frequently, the introns are shortened. The constructs are transfected into eukaryotic cells and usually analyzed by RT-PCR13,14. This approach has been extensively used to map regulatory splice sites and trans-acting factors in co-transfection experiments13,15,16,17,18. In addition, the generation of protein-expressing minigenes allowed for screening of substances that change alternative splicing19,20.
The method has been applied to circular RNAs. Currently, at least 12 minigene backbones have been described in the literature and are summarized in Table 1. With the exception of the tRNA based expression system21,22, they are all dependent on polymerase II promoters. Here, we describe a method to generate human reporter minigenes to determine cis and trans-acting factors involved in the generation of circular RNAs. An overview of the method using sequences of a published reporter gene23 is shown in Figure 1.

<table><tbody><tr><td>(PEI) Hydrochloride</td><td>Polysciences</td><td>24765-1</td><td></td></tr><tr><td>Builder tool</td><td>NEB</td><td></td><td>&lt;a href=&quot;https://nebuilder.neb.com/#!/&quot; target=&quot;_blank&quot;&gt;https://nebuilder.neb.com/#!/&lt;/a&gt;</td></tr><tr><td>Dark Reader Transilluminator.</td><td>Clare Chemical Research</td><td></td><td></td></tr><tr><td>Enzymatic DNA assembly kit</td><td>NEB</td><td>E2621S</td><td></td></tr><tr><td>Gel and PCR cleanup kit</td><td>Promega</td><td>A9282</td><td></td></tr><tr><td>Glyco Blue</td><td>Thermo Fisher</td><td>AM9516</td><td></td></tr><tr><td>pcDNA3.1 cloning site</td><td>Polycloning site</td><td></td><td>&lt;a href=&quot;https://assets.thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_1_man.pdf&quot; target=&quot;_blank&quot;&gt;https://assets.thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_1_man.pdf&lt;/a&gt;</td></tr><tr><td>Polymerase 1</td><td>NEB</td><td>M0491L</td><td>Q5 DNA polymerase</td></tr><tr><td>Polymerase 2</td><td>Biorad</td><td>1725310</td><td>Long range polymerase (NEB), iproof (BioRad)</td></tr><tr><td>Polymerase 2</td><td>Qiagen</td><td>206402</td><td>Qiagen long range polymerase kit</td></tr><tr><td>Reverse Transcriptase</td><td>Thermo Fisher</td><td>18080044</td><td></td></tr><tr><td>RNA isolation kit</td><td>Life Technologies</td><td>12183025</td><td>Ambion by Life Technologies</td></tr><tr><td>RNAse R</td><td>Lucigen</td><td>RNR07250</td><td>Epicenter/Lucigen</td></tr><tr><td>Stable competent cells</td><td>NEB</td><td>C3040H</td><td>NEB stable cells</td></tr><tr><td>Standard cloning bacteria</td><td>NEB</td><td>C2988J</td><td>NEB5-alpha competent</td></tr><tr><td>Web tool to design primers</td><td>NEB</td><td></td><td>&lt;a href=&quot;https://nebuilder.neb.com/#!/&quot; target=&quot;_blank&quot;&gt;https://nebuilder.neb.com/#!/&lt;/a&gt;</td></tr><tr><td>Web-based temperature calculations</td><td>NEB</td><td></td><td>&lt;a href=&quot;https://tmcalculator.neb.com/#!/main&quot; target=&quot;_blank&quot;&gt;https://tmcalculator.neb.com/#!/main&lt;/a&gt;</td></tr></tbody></table>

use of alu element containing minigenes to analyze circular rnas

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Reporter genes allow determination of regulatory factors that influence circular RNA formation. However, these reporter genes are large and contain repetitive elements that often make DNA constructs unstable. Due to their large size, it is often necessary to delete parts of the introns, which is achieved by amplifying genomic pieces containing the exons and smaller flanking intronic parts. These DNA pieces are enzymatically assembled, allowing construction without restriction enzymes.
The exam...

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Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

use-of-alu-element-containing-minigenes-to-analyze-circular-rnas

Research

JoVE Journal

Biochemistry

7.3K Views.  University of Kentucky. Dit protocol is belangrijk omdat het de gebruiker in staat stelt om een genomisch expressiesysteem te genereren dat alternatieve splicing kan ondergaan en in dit geval circulaire RNAs kan vormen die kunnen worden getest op hun functie en ziekteassociatie. Het belangrijkste voordeel van deze techniek is dat het de weg zal effenen voor anderen om dit protocol te gebruiken in de moleculaire biologie en klonen bij het bestuderen van alternatieve splicing in circulaire RNA formatie en functie. Mij...

Video: Use of Alu Element Containing Minigenes to Analyze Circular RNAs

RNA Next-Generation Sequencing and a Bioinformatics Pipeline to Identify Expressed LINE-1s at the Locus-Specific Level

Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and mutagenesis. Understanding the expression patterns of L1 loci at the individual level will lend to the understanding of the biology of this mutagenic element. This autonomous element makes up a significant portion of the human genome with over 500,000 copies, though 99% are truncated and defective. However, their abundance and dominant number of defective copies make it challenging to identify authentically expressed L1s from L1-related sequences expressed as part of other genes. It is also challenging to identify which specific L1 locus is expressed due to the repetitive nature of the elements. Overcoming these challenges, we present an RNA-Seq bioinformatic approach to identify L1 expression at the locus specific level. In summary, we collect cytoplasmic RNA, select for polyadenylated transcripts, and utilize strand-specific RNA-Seq analyses to uniquely map reads to L1 loci in the human reference genome. We visually curate each L1 locus with uniquely mapped reads to confirm transcription from its own promoter and adjust mapped transcript reads to account for mappability of each individual L1 locus. This approach was applied to a prostate tumor cell line, DU145, to demonstrate the ability of this protocol to detect expression from a small number of the full-length L1 elements.

Here, we present a bioinformatic approach and analyses to identify LINE-1 expression at the locus specific level.

Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and ...

Genetics

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.

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 ...

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the others contain 5&#39; monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5&#39; triphosphate. However, the triphosphate at primary 5&#39; termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5&#39; polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5&#39; triphosphate.
In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5&#39; polyphosphatase, which converts 5&#39; triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5&#39; polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5&#39; end and is insensitive to 5&#39; polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot.
By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.

We present a circular RT-PCR-based strategy by combining circular RT-PCR, quantitative RT-PCR, RNA 5&#39; polyphosphatase-treatment, and Northern blot. This protocol includes a normalization step to minimize the influence of unstable 5&#39; triphosphate, and it is suitable for discriminating and mapping the primary and processed transcripts stably accumulated in maize mitochondrion.

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the ...

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.

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.

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 ...

R-Loop Analysis by Dot-Blot

The three-stranded nucleic acid structure, R-loop, is increasingly recognized for its role in gene regulation. Initially, R-loops were thought to be the by-products of transcription; but recent findings of fewer R-loops in diseased cells made it clear that R-loops have functional roles in a variety of human cells. Next, it is critical to understand the roles of R-loops and how cells balance their abundance. A challenge in the field is the quantitation of R-loops since much of the work relies on the S9.6 monoclonal antibody whose specificity for RNA-DNA hybrids has been questioned. Here, we use dot-blots with the S9.6 antibody to quantify R-loops and show the sensitivity and specificity of this assay with RNase H, RNase T1, and RNase III that cleave RNA-DNA hybrids, single-stranded RNA, and double-stranded RNA, respectively. This method is highly reproducible, uses general laboratory equipment and reagents, and provides results within two days. This assay can be used in research and clinical settings to quantify R-loops and assess the effect of mutations in genes such as senataxin on R-loop abundance.

This protocol details a simple method that quantifies R-loop, a three-stranded nucleic acid structure that comprises of an RNA-DNA hybrid and a displaced DNA strand.

The three-stranded nucleic acid structure, R-loop, is increasingly recognized for its role in gene regulation. Initially, R-loops were thought to be the ...

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

Cancer Research

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

An easy-to-use RNA pull-down protocol is designed for the identification of RNAs engaged in direct RNA/RNA interaction with a long non-coding RNA. The protocol uses psoralen as a fixative to cross-link only RNA/RNA interactions and provides the whole direct RNA interactome of a long non-coding RNA when coupled with RNA sequencing.

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome ...

Analyzing tRNA Dynamics Using AQRNA-seq

AQRNA-seq for Quantifying Small RNAs

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification of the pool of small RNAs. The AQRNA-seq library preparation procedure described here involves the use of custom-designed sequencing linkers and a step for reducing methylation RNA modifications that block reverse transcription processivity, which results in an increased yield of full-length cDNAs. In addition, a detailed implementation of the accompanying bioinformatics pipeline is presented. This demonstration of AQRNA-seq was conducted through a quantitative analysis of the 45 tRNAs in Mycobacterium bovis BCG harvested on 5 selected days across a 20-day time course of nutrient deprivation and 6 days of resuscitation. Ongoing efforts to improve the efficiency and rigor of AQRNA-seq will also be discussed here. This includes exploring methods to obviate gel purification for mitigating primer dimer issues after PCR amplification and to increase the proportion of full-length reads to enable more accurate read mapping. Future enhancements to AQRNA-seq will be focused on facilitating automation and high-throughput implementation of this technology for quantifying all small RNA species in cell and tissue samples from diverse organisms.

Author Spotlight: AQRNA-seq Role in Mapping Small RNAs and Unraveling Protein Translation Mechanisms

Absolute quantification RNA sequencing (AQRNA-seq) is a technology developed to quantify the landscape of all small RNAs in biological mixtures. Here, both the library preparation and data processing steps of AQRNA-seq are demonstrated, quantifying changes in the transfer RNA (tRNA) pool in Mycobacterium bovis BCG during starvation-induced dormancy.

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate ...

Quantification of Circular RNAs Using Digital Droplet PCR

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 water-in-oil droplets, and the PCR occurs in the individual droplets. The dd-PCR has several advantages over conventional real-time qPCR, including increased accuracy in detecting low-abundance targets, omitting reference genes for quantification, eliminating technical replicates for samples, and showing high resilience to inhibitors in the samples. Recently, dd-PCR has become one of the most popular methods for accurately quantifying target DNA or RNA for gene expression analysis and diagnostics. Circular RNAs (circRNAs) are a large family of recently discovered covalently closed RNA molecules lacking 5' and 3' ends. They have been shown to regulate gene expression by acting as sponges for RNA-binding proteins and microRNAs. Furthermore, circRNAs are secreted into body fluids, and their resistance to exonucleases makes them serve as biomarkers for disease diagnosis. This article aims to show how to perform divergent primer design, RNA extraction, cDNA synthesis, and dd-PCR analysis to accurately quantify specific circular RNA (circRNA) levels in cells. In conclusion, we demonstrate the precise quantification of circRNAs using dd-PCR.

This protocol describes the detailed method of digital droplet PCR (dd-PCR) for precise quantification of circular RNA (circRNA) levels in cells using divergent primers.

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 ...

Biology

In Silico Identification and Characterization of circRNAs During Host-Pathogen Interactions

Circular RNAs (circRNAs) are a class of non-coding RNAs that are formed via back-splicing. These circRNAs are predominantly studied for their roles as regulators of various biological processes. Notably, emerging evidence demonstrates that host circRNAs can be differentially expressed (DE) upon infection with&#160;pathogens (e.g., influenza and coronaviruses), suggesting a role for circRNAs in regulating host innate immune responses. However, investigations on the role of circRNAs during pathogenic infections are limited by the knowledge and skills required to carry out the necessary bioinformatic analysis to identify DE circRNAs from RNA sequencing (RNA-seq) data. Bioinformatics prediction and identification of circRNAs is crucial before any verification, and functional studies using costly and time-consuming wet-lab techniques. To solve this issue, a step-by-step protocol of in silico prediction and characterization of circRNAs using RNA-seq data is provided in this manuscript. The protocol can be divided into four steps: 1) Prediction and quantification of DE circRNAs via the CIRIquant pipeline; 2) Annotation via circBase and characterization of DE circRNAs; 3) CircRNA-miRNA interaction prediction through Circr pipeline; 4) functional enrichment analysis of circRNA parental genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). This pipeline will be useful in driving future in vitro and in vivo research to further unravel the role of circRNAs in host-pathogen interactions.

In Silico Identification and Characterization of circRNAs During Host-Pathogen Interactions

The protocol submitted here explains the complete in silico pipeline needed to predict and functionally characterize circRNAs from RNA-sequencing transcriptome data studying host-pathogen interactions.

Circular RNAs (circRNAs) are a class of non-coding RNAs that are formed via back-splicing. These circRNAs are predominantly studied for their roles as ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

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