Size-selected fraction of sample 13
Total RNA was assessed for purity and size integrity using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano Chip (Agilent Technologies, USA). Using the Clontech SMARTer PCR cDNA Synthesis Kit (Takara Bio USA Inc), RNA was synthesized to cDNA. Briefly the CDS Primer IIA was first annealed to the polyA+ tail of transcripts, followed by first-strand synthesis with SMARTScribe Reverse Transcriptase. The first-strand product was subsequently used for large-scale PCR with PrimeSTAR GXL DNA Polymerase. One portion of the amplified cDNA product was used directly to construct a non-size selected SMRTbell library. In parallel, a second portion of the amplified cDNA product was first size selected using 0.75% Cassette on the Blue Pippin (SAGE Science, USA) to increase the yield of transcripts greater than 4 kb. SMRTbell libraries were constructed for both the non-size and size selected fractions using the SMRTbell Template Prep Kit 1.0 (PacificBiosciences, USA). DNA damage was first repaired and ends were prepared for ligation. The SMRTbell adapter was then ligated to the DNA, and the libraries were then purified with Ampure XP magnetic beads. After SMRTbell library construction, the Sequel sequencing primer was annealed and Sequel polymerase was bound to the libraries using the Sequel Binding Kit (Pacific Biosciences, USA). Ampure PB magnetic beads were used to remove unbound polymerase prior to sequencing. The non-size selected SMRTbell library and the size selected SMRTbell library were then sequenced on separate SMRT cells on the PacBio Sequel using a 20-hour data collection time.