Bluepippin pippinht system

A HiFi SMRTbell library was constructed using the SMRTbell Express Template Prep Kit v2.0 (Pacific Biosciences—PacBio, Menlo Park, California, Cat. #100-938-900) according to the manufacturer’s instructions. HMW gDNA was sheared to a target DNA size distribution between 15 and 20 kb. The sheared gDNA was concentrated using 0.45× of AMPure PB beads (PacBio, Cat. #100-265-900) for the removal of single-strand overhangs at 37 °C for 15 min, followed by further enzymatic steps of DNA damage repair at 37 °C for 30 min, end repair and A-tailing at 20 °C for 10 min and 65 °C for 30 min, ligation of overhang adapter v3 at 20 °C for 60 min and 65 °C for 10 min to inactivate the ligase, and nuclease treatment at 37 °C for 1 h. The SMRTbell library was purified and concentrated with 0.45× AMPure PB beads (PacBio, Cat. #100-265-900) for size selection using the BluePippin/PippinHT system (Sage Science, Beverly, Massachusetts; Cat. #BLF7510/HPE7510) to collect fragments greater than 7 to 9 kb. The 15 to 20 kb average HiFi SMRTbell library was sequenced at University of California Davis DNA Technologies Core (Davis, California) using 2 8M SMRT cells, Sequel II sequencing chemistry 2.0, and 30-h movies each on a PacBio Sequel II sequencer.

The HiFi SMRTbell library was constructed using the SMRTbell Express Template Prep Kit v2.0 (PacBio, Cat. #100-938-900) according to the manufacturer’s instructions. HMW gDNA was sheared to a target DNA size distribution between 15 and 20 kb. The sheared gDNA was concentrated using 0.45× of AMPure PB beads (PacBio, Cat. #100-265-900) for the removal of single-strand overhangs at 37 °C for 15 min, followed by further enzymatic steps of DNA damage repair at 37 °C for 30 min, end repair and A-tailing at 20 °C for 10 min and 65 °C for 30 min, ligation of overhang adapter v3 at 20 °C for 60 min and 65 °C for 10 min to inactivate the ligase, then nuclease treated at 37 °C for 1 h. The SMRTbell library was purified and concentrated with 0.45× AMPure PB beads (PacBio, Cat. #100-265-900) for size selection using the BluePippin/PippinHT system (Sage Science, MA; Cat. #BLF7510/HPE7510) to collect fragments greater than 7 to 9 kb. The 15 to 20 kb average HiFi SMRTbell library was sequenced at University of California Davis DNA Technologies Core (Davis, CA) using 2 8M SMRT cells, Sequel II sequencing chemistry 2.0, and 30-h movies each on a PacBio Sequel II sequencer.

The HiFi SMRTbell library was constructed using the SMRTbell Express Template Prep Kit v2.0 (PacBio, Cat. #100-938-900) according to the manufacturer’s instructions. HMW gDNA was sheared to a target DNA size distribution between 15 and 20 kb. The sheared gDNA was concentrated using 0.45 of AMPure PB beads (PacBio, Cat. #100-265-900) for the removal of single-strand overhangs at 37 °C for 15 min, followed by further enzymatic steps of DNA damage repair at 37 °C for 30 min, end repair and A-tailing at 20 °C for 10 min and 65 °C for 30 min, ligation of overhang adapter v3 at 20 °C for 60 min and 65 °C for 10 min to inactivate the ligase, then nuclease treated at 37 °C for 1 h. The SMRTbell library was purified and concentrated with 0.45 AMPure PB beads (PacBio, Cat. #100-265-900) for size selection using the BluePippin/PippinHT system (Sage Science, Beverly, MA; Cat #BLF7510/HPE7510) to collect fragments greater than 7 to 9 kb. The 15 to 20 kb average HiFi SMRTbell library was sequenced at UC Davis DNA Technologies Core (Davis, CA) using 3 8M SMRT cells, Sequel II sequencing chemistry 2.0, and 30-h movies each on a PacBio Sequel II sequencer.