PacBio IsoSeq of Facetotecta (library m54057_190926_040405) y-nauplii
All samples collected for PacBio library preparation and sequencing were collected at Sesoko Island. The first sample (Y1) was collected in October 2018 and contained ~600 individual y-larvae. The second sample (Y2) was collected in June 2019 and consisted of ~3,000 individual y-larvae. Total RNA templates of Y1 and Y2 were combined prior PacBio library preparation and sequencing due to low mRNA inputs (ng) of individual samples. Total RNA and DNA was isolated and purified from the pooled sample using the AllPrep TM DNA/RNA Kit (Qiagen). The quality and concentration of all extracted total RNA samples were analyzed using Bioanalyzer RNA 6000 Nano (Agilent Technologies) to measure the RNA integrity number (RIN). The extracted gDNA was purified using Agencourt AMPure XP (Beckman Coulter), following the protocol of the manufacturer. The concentration and quality of the purified gDNA were measured using a NanoDrop 2000 instrument (Thermo Fisher Scientific) and a Qubit fluorometric quantitation fluorometer (Thermo Fisher Scientific). The total (Y1 and Y2 combined) RNA extract of ~3,600 individually sampled y-larvae were used to construct a cDNA library. Full-length cDNAs were synthesized from high-quality total RNA (RIN > 9) with oligo-dT priming and amplified using the Clontech SMARTer PCR cDNA Synthesis Kit with PrimeSTAR GXL DNA Polymerase (Takara Bio, USA). The PCR products were purified with AMPure PB magnetic beads (PacBio), end-repaired, and constructed into an Iso-Seq library by ligating to the blunt-end adaptor of the SMRTBell Template Prep Kit 1.0 (Pacific Biosciences, USA). The PacBio raw reads were classified into CCS and non-CCS subreads by running the IsoSeq3 module in PacBio SMRT Analysis v6.0. IsoSeq v3 determines a CCS or subread sequence to be full length if the 5- and 3-primers and polyA tail signal were all present in the correct order. Read of Insert (ROI) was generated by using minimum 8 full passes from each zero-mode waveguide (ZMW). Reads that did not contain the 5 or 3 adapters, a poly-A/polyT tail or had read length < 100 nucleotides were removed. After clustering, the full-length transcripts were polished with predicted consensus accuracy 0.99, which were considered polished high-quality transcripts (IsoSeqs).