title: PacBio HiFi sequencing data for Diaphorina citri (APC01)
design: A whole insect (ACP01) was homogenized in 0.6 ml of homogenization buffer (10 mM Tris-HCL-pH 8.0, 25 mM EDTA) using TissueRuptor II (Qiagen Cat # 9002755) and an equal volume of lysis buffer (10 mM Tris, 25 mM EDTA, 200 mM NaCl, and 1% SDS) and proteinase K (100 g/ml) were added to the homogenate and incubated overnight at room temperature. The lysate was further treated with RNAse A (20 g/ml) at 37 C for 30 minutes and cleaned with equal volumes of phenol/chloroform using phase-lock gels (Quantabio, MA Cat # 2302830). The DNA in the cleaned lysate was precipitated by adding 0.4 and 3 volumes of 5M ammonium acetate and ice-cold ethanol, respectively, followed by centrifugation at 13000 x g for 10 minutes at room temperature. The DNA pellet was washed twice with 70% ethanol and resuspended in an elution buffer (10 mM Tris, pH 8.0). The DNA yield (128 ng) was quantified using the Quantus Fluorometer (QuantiFluor ONE dsDNA Dye assay; Promega, WI), and the size distribution of the DNA fragments was estimated using the Femto Pulse system (Genomic DNA 165 kb kit, Agilent, CA), where 53 % of the DNA fragments were found to be 30 kb or longer. The HiFi SMRTbell library was constructed using the SMRTbell gDNA Sample Amplification Kit (Pacific Biosciences, Menlo Park, CA; Cat. #101-980-000) and the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences; Cat. #100-938-900) according to the manufacturer's instructions. Approximately 10 kb sheared DNA by the Megaruptor 3 system (Diagenode, Belgium; Cat. #B06010003) was used for removal of single-strand overhangs at 37C for 15 minutes, DNA damage repair at 37C for 30 minutes, end repair and A-tailing at 20C for 30 minutes and 65C for 30 minutes, and ligation of amplification adapters at 20C for 60 minutes. To prepare for library amplification by PCR, the library was purified with ProNex beads (Promega, Madison, WI; Cat. # NG2002) for two PCR amplification conditions at 15 cycles each then another ProNex beads purification. Purified amplified DNA from both reactions were pooled in equal mass quantities for another round of enzymatic steps that included DNA repair, end repair/A-tailing, overhang adapter ligation, and purification with ProNex Beads. The PippinHT system (Sage Science, Beverly, MA; Cat # HPE7510) was used for SMRTbell library size selection to collect fragments between 6-10 kb. The 10  11 kb average HiFi SMRTbell library was sequenced at UC Davis DNA Technologies Core (Davis, CA) using one 8M SMRT cell, Sequel II sequencing chemistry 2.0, and 30-hour movies each on a PacBio Sequel II sequencer.