title: Pacbio Sequel II cell C01
design: 1) DNA Shearing: The SMRTbell library target size to construct depends on the goals of the project and the quality and quantity of the starting gDNA. The g-TUBE can be used to shear gDNA to 20 kb fragments for constructing 20 kb SMRTbell libraries.  2) Remove Single-Strand Overhangs: Exo was used to treat DNA for shearing long overhangs before DNA damage repair. 3) Repair DNA Damage: We use reagents included in the Template Prep Kit to repair abasic sites, nicks, thymine dimers, blocked 3-ends, oxidized guanines/pyrimidines, deaminated cytosine. 4) Repair Ends for Blunt-End Ligation: This step aims to polish ends of fragments suitable for ligation. T4 DNA Polymerase was used to fill in 5 overhangs and remove 3 overhangs. And T4 PNK was used to phosphorylates 5 hydroxyl group. 5) Blunt-End Ligation: During this step, SMRTbell hairpin adapters included in Template Prep Kit are ligated to repaired ends. 6) Purify Templates: Imperfect SMRTbell templates were removed with EXO (from 3-hydroxyl termini and nicks) and  (from 5-termini) treatment. AMPure PB Beads were used to concentrate and purify libraries then. 7) Size Selection with the BluePippin System: When constructing large insert SMRTbell libraries for whole genome sequencing of complex organisms, it is beneficial to remove small insert SMRTbell templates by performing size selection with the BluePippin System (which collects fragments above a size cut-off threshold). In this case, we do size selection using BluePippin System and set size cut-off threshold depending on the goals of the project. Then, AMPure PB Beads were used to concentrate and purify SMRTbell templates after size selection. 8) Anneal Sequencing Primer to SMRTbell Template: Sequencing primer annealed to both ends of the SMRTbell templates. 9) Bind Sequencing Polymerase to SMRTbell Template: For efficient loading into ZWMs, polymerase is bound to both ends of SMRTbell templates using Binding Kit. 10) Prepare for Sequencing: Use DNA Sequencing Reagent Kit and follow the manual to load libraries in SMRT Cells.