Micromonas pusilla full-length mRNA transcript consensus sequences
We extracted RNA from M. pusilla strain RCC834 (also known as CCMP1545). We prepared cDNA libraries and circularized the cDNA libraries via Gibson assembly using a short, pre-annealed double-stranded DNA splint overlapping the cDNA ends. To remove uncircularized molecules, we digested the reaction with ExoI, ExoII, and Lambda Exonuclease. We cleaned the reaction with SPRI beads at a 1:0.85 ratio.We used the clean, circularized library as a template for rolling circle amplification (RCA) with Phi29 polymerase and a random hexamer primer. We then debranched the Phi29 reaction using T7 endonuclease. We size-selected the resulting R2C2 library for DNA fragments larger than 3 kb using ProNex beads. We sequenced the multiplexed R2C2 library on an ONT PromethION R10.4 flow cell and basecalled reads with the Dorado basecaller using model dna_r10.4.1_e8.2_400bps_sup@v5.0.0. The resulting raw reads were converted into consensus reads and demultiplexed using C3POa (v3.2). Based on these demultiplexed consensus reads, Mandalorion (v4.6) was used to identify and quantify high-confidence isoforms.