PacBio HiFi sequencing data from Pisaster brevispinus: tube foot from individual M0D059179O
We constructed the PacBio HiFi library using the SMRTbell Express Template Prep Kit v2.0 according to the manufacturer's instructions. We sheared 13.1 ug of HMW gDNA to an average size distribution of ~18kb using Diagenode's Megaruptor 3 system. We quantified the sheared DNA using the Quantus Fluorometer and checked the size distribution using the Agilent Femto Pulse. We concentrated the sheared gDNA using 0.45X of AMPure PB beads followed by quantification using a Quantus Fluorometer. We used 6 ug of sheared, concentrated DNA as input for the removal of single-strand overhangs at 37 C for 15 minutes, followed by further enzymatic steps of DNA damage repair at 37 C for 30 minutes, end repair and A-tailing at 20 C for 10 minutes and 65 C for 30 minutes, ligation of overhang adapter v3 at 20 for 1 hour and 65 for 10 minutes to inactivate the ligase, and nuclease treatment of SMRTbell library at 37 for 1 hour to remove damaged or non-intact SMRTbell templates. We purified and concentrated the SMRTbell library with 0.8X Ampure PB beads for size selection using the BluePippin system. We purified the input of 3.2 ug purified SMRTbell library to load into the Blue Pippin 0.75% Agarose Cassette using cassette definition 0.75% DF Marer S1 3-10kb Improved Recovery for the run protocol. We collected fragments greater than 7 kb from the cassette elution well and purified and concentrated the size-selected SMRTbell library with 0.8X AMPure beads.  The 20.5 kb average HiFi SMRTbell library was sequenced using one 8M SMRT Cell, Sequel II sequencing chemistry 2.0, and 30-hour movies on a PacBio Sequel II sequencer.