Experimental design. (A) Total RNA from cardiac apex, extensor digitorum longus (EDL), and soleus muscles were extracted, and cDNA was generated using barcoded oligo(dT) primers. Next, 5–10 kb cDNAs from all muscles were size selected, pooled for library construction, and sequenced on the PacBio RSII. (B) PacBio's Iso-Seq bioinformatics pipeline. Raw reads are converted into circular consensus (CCS/HiFi) reads. HiFi reads are separated into full-length (FL) and non-FL reads. FL reads must have 5′ and 3′ primers and a poly(A) tail. FL reads are grouped by similarity (isoform), polished using non-FL reads to generate high quality transcript consensus reads, and aligned to the mouse genome. (Reprinted, with permission, from Pacific Biosciences.)
