Sequel smrt cell 1m v2

Genomic DNA were extracted from tender leaves of ‘Wongyo 3115’, using the CTAB method (Lee et al., 2020 (link)) and fragmented into 20 kb, using a g-TUBE (Covaris, USA). Furthermore, the fragments were purified, using AMpureXP bead purification system to remove the small fragments. After purification, the SMRTbell library was constructed, using SMRTbell™ Template Prep Kit 1.0 (PN 100-259-100), and the BluePippin Size selection system was employed to remove the small fragments for a large-insert library. Using Sequel Binding Kit (2.0), sequencing primer and DNA polymerase were bound to the SMRTbell library, and the complex was purified with SMRTbell Clean-up columns (SMRTbell® Clean Up Columns v2 Kit-Mag: PN 01-303-600). The MagBead Kit (Pacific Biosciences) was used to bind the library complex with MagBeads before sequencing. The polymerase-SMRTbell-adaptor complex was then loaded into zero-mode waveguides (ZMWs). The SMRTbell library was sequenced, using 25 SMRT cells (Pacific Biosciences, Sequel™ SMRT® Cell 1M v2) with Sequel Sequencing Kit (2.1), and 1 × 600-min movies were captured for each SMRT cell, using the Sequel (Pacific Biosciences)-sequencing platform. Finally, the resulting Sequel raw bam files were converted into subreads in the FASTA format, using the standard PacBio SMRT Link v10.1 software package.

The total RNA was isolated from the callus tissue, and the cDNA synthesis was carried out, using the SMARTer PCR cDNA Synthesis Kit (Clontech 634925) and PCR using PrimeSTAR GXL DNA Polymerase (Clontech R050A). Further purification was performed, using AMPure® PB Bead prior to the library construction. For the construction of SMRTbell library, 1-5 μg of pooled cDNA was prepared, using SMRTbell™ Template Prep Kit 1.0-SPv3 (PN 100-991-900). The SMRTbell library was sequenced, using SMRT cells per library (Pacific Biosciences, Sequel™ SMRT® Cell 1M v2). A total of four SMRT cells were sequenced, using the PacBio Sequel platform with 1,200 min of movie time.