PacBio HiFi sequencing data for Eriogonum infllatum: sample FZ781
High molecular weight (HMW) genomic DNA (gDNA) was isolated from 745 mg of leaf tissue (Sample I.D. FZ-781) using the cetyltrimethylammonium bromide (CTAB) method as described in Inglis et al., 2018, with several modifications: (i) we substituted 2-mercaptoethanol (1% v/v) with sodium metabisulfite (1% w/v) in both the sorbitol wash buffer and CTAB solution; (ii) we continued the wash steps of the tissue homogenate until obtaining a clear supernatant; (iii) we carried out the CTAB lysis step at 45C; and (iv) we performed the chloroform extraction step twice using ice-cold chloroform. We evaluated DNA purity (260/280 = 1.85 and 260/230 = 2.12) using absorbance ratios measured on a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, MA). The DNA yield (34 g total) was determined using the QuantiFluor ONE dsDNA Dye assay (Promega, WI). The size distribution of the HMW DNA was analyzed using the Femto Pulse system (Genomic DNA 165 kb kit, Agilent, CA), revealing that 70% of the total fragments were larger than 30 kb.  The HiFi SMRTbell library was constructed using the SMRTbell Express Template Prep Kit v2.0 (Pacific Biosciences, Menlo Park, CA; Cat. #100-938-900) according to the manufacturer's instructions. HMW gDNA was sheared to a target DNA size distribution between 15 kb  18 kb using Diagenodes Megaruptor 3 system (Diagenode, Belgium; cat. B06010003). The sheared gDNA was concentrated using 0.45X of AMPure PB beads (Pacific Biosciences, Menlo Park, CA; Cat. #100-265-900) for the removal of single-strand overhangs at 37C for 15 minutes, followed by further enzymatic steps of DNA damage repair at 37C for 30 minutes, end repair and A-tailing at 20C for 10 minutes and 65C for 30 minutes, and ligation of overhang adapters v3 at 20C for 60 minutes. The SMRTbell library was purified and concentrated with 1X AMPure PB beads for nuclease treatment at 37C for 30 minutes and size selection using the BluePippin/PippinHT system (Sage Science, Beverly, MA; Cat #BLF7510/HPE7510) to collect fragments greater than 7-9 kb. The 15  20 kb average HiFi SMRTbell library was sequenced at UC Davis DNA Technologies Core (Davis, CA) using three 8M SMRT cell, Sequel II sequencing chemistry 2.0, and 30-hour movies two on a PacBio Sequel II sequencer and one on a PacBio Sequel IIe sequencer
SUB15451857
CCGP_gDNA0057