Agilent 5200 fragment analyzer system

Total RNAs were extracted from liver and muscle tissue of mako and isolated using the RNeasy Plus Universal total RNA kits (Qiagen, Valencia, CA, USA) as per the manufacturer’s standard protocol. Following RNA isolation, the samples were concentrated using RNA Clean & Concentrator Kit (Zymo Research, Irvine CA, USA) and their purity was checked using the DeNovix DS-11 + spectrophotometer (DeNovix Inc., Wilmington, DE, USA). RNA concentration was measured using Qubit RNA Assay Kit in Qubit 3.0 Fluorometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The integrity of total RNA was assessed on the Agilent Fragment Analyzer 5200 system (Agilent Technologies, Santa Clara, CA, USA) using the High Sensitivity RNA Kit with an RNA Quality Number (RQN) criteria of RQN >7.0.

A total of 100 ng - 300 ng of RNA was input for cDNA synthesis and amplification using NEBNext Single Cell/Low Input cDNA Synthesis & amp, Amplification Module (New England BioLabsInc., Ipswich, MA, USA) as per the manufacturer’s standard protocol. PCR cycles of 10–15 were used to get sufficient quantities of cDNA for pacbio library preparations. Concentration and size profile of cDNA samples was assessed on the Agilent Fragment Analyzer 5200 system (Agilent Technologies, Santa Clara, CA, USA) using the High Sensitivity Large Fragment Kit.
Amplified cDNA samples were size selected using ProNex Size-Selective Purification System (Promega Corporation, Madison, WI, USA) as per the PacBio recommendation for standard length cDNA transcripts. Size selected cDNA was used to construct SMRTbell Iso-Seq libraries using Express Template Prep 2.0 (Pacific Biosciences, Menlo Park, CA, USA) as per the manufacturer’s Iso-Seq Express Template Preparation protocol. The quality of the Iso-Seq libraries were assessed using the Qubit 3.0 Fluorometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and the Agilent Femto Pulse System (Agilent Technologies, Santa Clara, CA, USA).