Supplementary Figure 2: Verification of circRNAs with RT-PCR and FISH.

(a) RT-PCR was performed with divergent primers corresponding to intron (primer set 1) or exon (primer set 2) sequences in circEIF3J and circPAIP2. PCR products show that introns were retained in circEIF3J and circPAIP2. (b) RT-PCR was performed with divergent primers corresponding to the 5’ exon and upstream intron closest to the 3’ exon. PCR products show that at least the upstream intron closest to the 3’ exon was retained in all of the other 13 circRNAs. (c) Separate channels are shown for FISH images for circEIF3J and circPAIP2 in Fig. 1f. From these and subsequent FISH images, it is clear that circEIF3J and circPAIP2 are localized in the nucleus, but both also have some cellular heterogeneity in the nuclear distribution and perhaps copy number. (d) Separate channels are shown for the FISH images of EIF3J and PAIP2 mRNA in Fig. 1f. (e) Double FISH of the parental gene loci and a downstream intron of the indicated circRNA in the parental gene pre-mRNA. Essentially no FISH signal of the downstream intron was observed for the downstream intron. For EIF3J, only one (indicated with a white arrowhead) out of 38 cells imaged showed a positive FISH signal for the downstream intron (n=38 cells for EIF3J, and 30 cells for PAIP2). (f) FISH Images of circRNAs with or without siRNA knockdown of the indicated circRNA. (g) Dual FISH of circRNAs and mRNA of the parental genes after treatment with RNase R. No FISH signal was detected for the mRNA. For all FISH images for circRNAs, junction probes were used unless specified. All FISH images were with HEK293 cells.