title: PacBio HiFi sequencing data for Orcuttia inaequalis (DT0271)
design: High molecular weight (HMW) genomic DNA (gDNA) was isolated from 62 mg of young leaf and floral tissues using the cetyltrimethylammonium bromide (CTAB) method as described by Inglis et al. (2018), with the following modifications: (i) we replaced 2-mercaptoethanol (1% v/v) with sodium metabisulfite (1% w/v) in both the sorbitol wash buffer and the CTAB solution; (ii) we continued washing the tissue homogenate until a clear supernatant was obtained; (iii) we conducted the CTAB lysis step at 45C; and (iv) we performed the chloroform extraction step twice using ice-cold chloroform.  We removed the majority of shorter DNA fragments (<10 kb) using the Short Read Eliminator kit (PacBio, Ca), following the manufacturers instructions.  We evaluated DNA purity (260/280 = 1.84 and 260/230 = 2.42) using absorbance ratios measured on a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, MA). The final DNA yield (5.6 g total) was quantified using QuantiFluor ONE dsDNA Dye assay (Promega, WI). The size distribution of the HMW DNA was analyzed with the Femto Pulse system (Genomic DNA 165 kb kit, Agilent, CA), revealing that 97 % of the total fragments were larger than 10 kb. The HiFi SMRTbell library was constructed using the SMRTbell prep kit 3.0 (Pacific Biosciences, Menlo Park, CA; Cat. #102-182-700) according to the manufacturer's instructions. HMW gDNA was sheared to a target DNA size distribution between 15-18 kb using Diagenodes Megaruptor 3 system (Diagenode, Belgium; cat. B06010003). The sheared gDNA was concentrated using 1X of SMRTbell cleanup beads provided in the SMRTbell prep kit 3.0 for the repair and a-tailing incubation at 37C for 30 minutes and 65C for 5 minutes, followed by ligation of overhang adapters at 20C for 30 minutes, clean-up using 1X SMRTbell cleanup beads, and nuclease treatment at 37C for 15 minutes. The SMRTbell library was size selected using 3.1X of 35% v/v diluted AMPure PB beads (Pacific Biosciences, Menlo Park, CA; Cat. #100-265-900) to progressively remove SMRTbell templates <5kb.  The 15  18 kb average HiFi SMRTbell library was sequenced at UC Davis DNA Technologies Core (Davis, CA) using two 8M SMRT cells (Pacific Biosciences, Menlo Park, CA; Cat #101-389-001), Sequel II sequencing chemistry 2.0, and 30-hour movies each on a PacBio Sequel IIe sequencer.