Iso-Seq of Peromyscus maniculatus brain, liver, testes, spinal cord, VNO and MOE
Six tissues were pooled for Iso-Seq experiment: liver, brain, testes, spinal cord, vomeronasal organ (VNO) and main olfactory epithelium (MOE). Liver, brain and testes tissues were equally represented in the Iso-Seq data. Spinal cord is represented at 1/2 the coverage of liver, and VNO & MOE each at 1/4 the coverage of liver. Total RNA was extracted from fresh frozen tissue samples using Qiagen RNeasy Plus Universal mini kit following manufacturers instructions (Qiagen, Hilden, Germany). RNA samples were quantified using Qubit 2.0 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 SMARTer PCR cDNA synthesis Kit (Clontech, Mountain View, CA, USA). The library for PacBio Sequel was constructed using SMRTbell Express Template Prep Kit 3.0 (PacBio, Menlo Park, CA, USA).The library was bound to polymerase using the Sequel Binding Kit (PacBio) and loaded onto PacBio Sequel using the MagBead Kit V2 (PacBio). Sequencing was performed on 1 PacBio Sequel II SMRT Cell (24-hour movie time). After sequencing, CCS reads were obtained using the CCS algorithm within PacBio ccs v4.2.0. The algorithm was run using the default parameters. Lima was used to remove cDNA primers and iso-seq refine used to remove polyA tails and artificial concatemers.