Sequel II IsoSeq of Sepia officinalis
RNA was isolated from 5-10 mg of flash-frozen tissue (brain, mantle/epidermis, arm/tentacle). First, tissue samples were homogenized in TRIzol using a homogenizer (VDI12/S12N-5S, VWR, Germany). RNA was isolated and DNAseI treated according to the Direct-zol RNA Mini prep kit from ZymoResearch (R2050). RNA was quantified by spectrophotometry (Nanodrop, Thermo Scientific, USA) and its quality was assessed with a RNA 6000 Nanochip on Bioanalyzer (Agilent Technologies, Germany).The Iso-Seq library was prepared and sequenced at the Sequencing facility of the MPI for Plant Research, using a method targeting the 5 cap and poly-A tail of mRNAs, described in detail in4. Briefly, mRNA was pooled from the 8 tissue samples and cDNA was synthesized using the TeloPrime Full-Length cDNA Amplification Kit V2 (Lexogen, Cat Nr: 013.08 or 013.24). The sequencing library was prepared with the SMRTbell express template prep kit 2.0 (PacBio, Cat. no. 100-938-900).The library was sequenced on 2 SMRT cells on the PacBio Sequel II system using the Sequel II binding kit 2.1 (101-843-000), Sequel II sequencing kit 2.0 (101-820-200) and SMRT Cell 8M tray (101-389-001).