IsoSeq3 sequencing of the transcriptome associated with a pool of different tissues from Dissostichus eleginoides
RNA was extracted from individual tissues using the Direct-zol RNA Miniprep Plus kit (Zymo Research) according to the manufacturers instructions. Briefly, 50 mg of the toothfish tissue was ground to a powder with liquid nitrogen in a precooled mortar and pestle. and then lysed in 600 l of TRI Reagent. The lysed sample was transferred to a clean 1.5 microtube and an equal volume of absolute ethanol added. The sample was thoroughly mixed and 700 l transferred to the Zymo-Spin IIICG column and centrifuged at 13,000g for 30 sec. The flow through was discarded and the column treated with DNase 1 prior to washing with 400 l of Direct-zol RNA prewash, followed by a wash with 700 l RNA wash buffer. The RNA was then eluted in 100 l DNase/RNase free water and stored at -80C.  RNA quality was assessed via Tapestation using the RNA Analysis ScreenTape System (Agilent). SMRTBell libraries were generated following the Iso-Seq Express protocol (https://www.pacb.com/wp-content/uploads/Procedure-Checklist-Iso-Seq-Express-Template-Preparation-for-Sequel-and-Sequel-II-Systems.pdf). The cDNA libraries were cleaned up using the standard purification method with ProNEX beads. A total of 3 SMRT cells were run on the PacBio Sequel.   Following sequencing PacBio data was processed using version 9.0 of the SMRT Link software from Pacific Biosciences. Version 3.4.0 of the IsoSeq protocal was used for consensus sequence generation, orientation, removal of primers, polyA tails and splitting up of concatemers, resulting in a set of 'FL Non-Concatemer (FLNC) reads'.