IsoSeq of Phyllotreta striolata: pooled adults
Adult beetles were pooled in a 1.5 ml tube, ground, and total RNA was extracted using the standard TRIzol Reagent protocol (Invitrogen), and then further cleaned using an Illustra RNAspin Mini Kit with DNase treatment (GE Healthcare Life Sciences) to remove any salts and potential contaminants. Total RNA concentration was quantified using a NanoDrop One spectrophotometer (Thermo Scientific) and quality assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Total RNA (1 g) from each replicate and tissue type was sent to Azenta Genewiz (South Plainfield, NJ), who generated the IsoSeq library and performed the sequencing as follows: RNA samples were quantified using Qubit 4.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrities were checked with TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). For Iso-Seq library construction, 300 ng of total RNA was converted into amplified full-length cDNA using the NEBNext® Single Cell/Low Input RNA Library Prep Kit (NEB) The library for PacBio Sequel was constructed using SMRTbell Express Template Prep Kit 3.0 (PacBio, Menlo Park, CA, USA). The final library was bound to polymerase and loaded onto PacBio Revio using the Revio Polymerase Kit (PacBio: 102-739-100). Movie Acquisition Time was set to 24hr. Iso-Seq barcode demultiplexing was performed. The CCS reads were classified into full-length and non-full-length reads based on the presence of cDNA primers and polyA tails in the reads. If both primers and polyAs were seen in a read then it was classified as a full-length read, otherwise the read was treated as a non-full-length read.