*4.2. RNA Extraction and CircRT-PCR*

To promote RNA production, *P. duplex* cultures were incubated in a shaking incubator (50 rpm) at constant temperature (28 ◦C) and artificial light (500 μE m−<sup>2</sup> s−1) for several hours prior to extraction. Cells were pelleted using a Beckman-Coulter Avanti JXN-30 centrifuge, and pellets were resuspended in Qiagen's RNeasy extraction buffer (Germantown, MD, USA) and transferred to a bead beater tube. Cells were lysed by bead beating on a vortexer for five minutes. The lysate was then taken through the remaining Qiagen RNeasy protocol with the optional DNase step. *C. vulgaris* tissues were flash frozen in liquid nitrogen and ground with a mortar and pestle followed by RNA extraction using a Qiagen RNeasy kit, as described in Cahoon et al. [59]. All RNA samples were quantified using a NanoDrop Lite (Thermo-Fisher, Waltham, MA, USA) and stored at −80 ◦C.

Primers for cDNA synthesis and PCR (Supplemental Table S1) were designed using Primer3 (https://primer3.org) and synthesized by Integrated DNA Technologies (Coralville, IA, USA). RT-PCR was completed as described in Meade et al. [91]. Briefly, RNAs were artificially circularized using 2 μg of total RNA and T4 RNA ligase (New England Biolabs, Ipswich, MA, USA). cDNAs were synthesized from the circularized RNAs using the R1 primers and MMLV Reverse Transcriptase (Promega, Madison, WI, USA). These cDNAs were then used as template, along with the R1 and L1 primers to PCR amplify the 3- –5 junctions of each transcript using Phusion DNA polymerase (ThermoFisher, Waltham, MA, USA). The products of these PCR reactions were diluted 10-fold and used as template for a second round of PCR using primers R2 and L2. This process was completed twice with RNA extracted at two different times to represent independent replicates. Naturally circularized mRNAs were detected producing cDNA directly from 2 μg of total RNA without T4 ligase treatment followed by two rounds of PCR as described above. The naturally circularized mRNA process was also completed twice.

## *4.3. Sequencing and Analysis*

The independent replicates of the secondary PCR products for both *P. duplex and C. vulgaris* were deep sequenced, separately, using Genewiz's Amplicon EZ Illumina MiSeq service (South Plainfield, NJ, USA). Sequences were analyzed using Geneious Prime software (Biomatters, Auckland, New Zealand). Initially, sequences were matched to each gene using the Map-to-Reference function. These sequences were visually inspected, for 3- poly-nucleotide additions, and sequence motifs. The 3- –5 junction sites were identified using NCBI's BLAST align two or more sequences feature (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

For PacBio Iso-Seq, ~1 g of total RNA from each species was polyadenylated using Lucigen's (Middleton, WI, USA) Poly(A) Polymerase Tailing kit according to the manufacturer's protocol. The tailed samples were cleaned using Qiagen's RNeasy kit. Samples were shipped to GeneWiz on dry ice for PacBio library preparation and sequencing. This process was completed once. Reads were aligned to the *P. duplex* and *C. vulgaris* chondriomes with Geneious Prime software using the Map-to-Reference function.

Logo plots were generated using the web-based service https://weblogo.berkeley. edu/logo.cgi. RNA secondary structures were predicted using RNAfold (http://rna.tbi. univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi).
