Iso-Seq of M. javanica VW4
cDNA synthesis was prepared using NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module kit (New England Biolabs Inc., Ipswich, MA; Cat #E6421L) according to manufacturer's instructions with a slight modification to the PCR cycles of 15 cycles. Amplified cDNA samples were purified using 0.86X of ProNex beads (Promega, Madison, WI; Cat #NG2003) for equal molar pooling of barcoded cDNA. The pooled cDNA was constructed into the SMRTbell Express Template Prep Kit v2.0 (Pacific Biosciences, Menlo Park, CA; Cat. #100-938-900) according to the manufacturer's instructions. The enzymatic steps included DNA damage repair at 37 C for 30 minutes, end repair and A-tailing at 20 C for 30 minutes and 65 C for 30 minutes, and ligation of adapters v3 at 20 C for 60 minutes. The SMRTbell library was purified with 1X ProNex beads. The IsoSeq SMRTbell library was sequenced at UC Davis DNA Technologies Core (Davis, CA) using one SMRT Cell 8M Tray (Pacific Biosciences, Menlo Park, CA; Cat #101-389-001), Sequel II sequencing chemistry 2.0, and 30-hour movies each on a PacBio Sequel II sequencer.