title: PB Transcriptome for Mus musculus: heart
design: Total RNA was extracted by grinding tissue in TRIzol reagent (TIANGEN)(Animal)/CTAB-LiCl method (Plant)/RNeasy Mini Kit(Qiagen,CA,USA)(cells) on dry ice processed following the protocol provided by the manufacyurer.Quality and quantity of total RNA were evaluated using a NanoDrop One UVVis spectrophotometer (ThermoFisher Scientific, USA) and Qubit 4.0 Fluorometer (ThermoFisher Scientific, USA), respectively.The integrity of total RNA was assessed via a Bioanalyzer 2100 System run using the RNA 6000 Nano Kit (Agilent,Technologies,USA),and only RNAs with RNA integrity numbers (RIN) above 8.0 were used for subsequent treatments and sequencing.The total RNA was used to construct Kinnex full-length RNA library using the Iso-Seq express 2.0 kit (PacBio 103-071-500) and Kinnex full-length RNA kit(PacBio103-072-000).Following the Kinnex full-length RNA library prep protocol. First, the first-strand cDNA was synthesized using Poly(dT) primer and Template Switch Oligo (TSO), and then the first-strand cDNA products were PCR-amplified and barcoded using barcoded Iso-Seq primers to generate the second-strand cDNA.The barcoded full-length cDNA products were pooled for input into Kinnex full-length RNA library construction,Using the Kinnex PCR 8-fold kit(PacBio 103-071-600) to perform 8 parallel Kinnex PCR reactions and generating DNA fragments containing orientationspecific Kinnex segmentation sequences. Kinnex PCR products were pooled, and then were treated with Kinnex enzyme, ligase, and barcoded Kinnex terminal adapters(PacBio 103-071-800)to assemble cDNA transcripts into a linear, ordered array. Final Kinnex full-length RNA library was obtained by performing nuclease treatment and final SMRTbell bead cleanup to remove incomplete SMRTbell template arrays.Kinnex full-length RNA library was sequenced on PacBio Revio platform with Kinnex sequencing primer and Revio polymerase kit (PacBio102-317600) in Grandomics Biosciences Co., Ltd (Wuhan, China).