PacBio RS II sinlge-end Sequencing of KAS22054414 (KAE22249800)
Construction of reference genome of 'Baekdansim' and development of cold resistance markers
Genomic DNA gDNA of high quality and high molecular weight was extracted from H. syriacus Baekdansim using a protocol described in a previous study1. PacBio genomic DNA library prep was performed according to the manufacturer's instructions https://www.pacb.com/support/documentation/. Purified gDNA was used for library construction by the SMRTbell xpress Template Prep Kit Pacific Biosciences, Cat. No. 101-357-000. In brief, gDNA was mechanically sheared to an average size of 20 kb using a Covaris g-TUBE device Part No. 520079. In total, 5 microgram sheared gDNA was damage-repaired and end-repaired using polishing enzymes. Blunt-end adapter ligation was used to create the SMRTbell emplate. Adapter dimers and contaminants were removed using the AMPure XP bead purification system Beckman Coulter, Cat. No. A63882. A BluePippin size selection system Sage Science, Cat. No. BLU0001 was used to size select the SMRTbell emplate and enrich for fragments >15 kb. Sequencing primer v4 was annealed to the SMRTbell emplate, and DNA polymerase/template complex was created using the Sequel Binding Kit 2.1 Pacific Biosciences, Cat. No. 101-365-900. An additional AMPure XP purification step was performed to remove excess primer and polymerase prior to sequencing. The library was sequenced on a Sequel instrument using SMRT Cell 1M v2 Pacific Biosciences, taking one movie of 10 hours per cell with the Sequel Sequencing Kit 2.0 Pacific Biosciences.