PacBio HiFi sequencing data for Aptostichus stephencolberti: sample BMEA0104753
Whole spider was flash frozen in liquid nitrogen (LN2). Half of the cephalothorax and front legs of the frozen sample were homogenized by grinding in mortar and pestle in the presence of LN2. Homogenized tissue was lysed with 2ml of lysis buffer containing 100 mM NaCl, 10 mM Tris-HCL-pH 8.0, 25 mM EDTA, 0.5% SDS and 100 g/ml proteinase K overnight at room temperature. Lysate was treated with 20 g/ml RNase at 370C for 30 minutes. The lysate was cleaned with equal volumes of phenol/chloroform using phase-lock gels (Quantabio Cat # 2302830). The DNA was precipitated by adding 0.4X volume of 5M ammonium acetate and 3X volume of ice-cold ethanol. The DNA pellet was washed twice with 70% ethanol and resuspended in an elution buffer (10mM Tris, pH 8.0). Purity of gDNA was accessed using NanoDrop ND-1000 spectrophotometer and 260/280 ratio of 1.82 and 260/230 of 2.13 were observed. DNA yield was 30 g as quantified using Qbit 2.0 Fluorometer (Thermo Fisher Scientific, MA). The integrity of the high-molecular-weight DNA was verified on a Pippin Pulse gel electrophoresis system (Sage Sciences, Beverly, MA) where majority of the DNA was observed in fragments > 120Kb.  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  20 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) to remove single-strand overhangs at 37C for 15 minutes.  This was followed by enzymatic steps:  DNA damage repair at 37C for 30 minutes, end repair and A-tailing at 20C for 10 minutes and 65C for 30 minutes, ligation of overhang adapters v3 at 20C for 60 minutes and 65C for 10 minutes to inactivate the ligase, and finally, nuclease treatment at 37C for 1 hour. The SMRTbell library was purified and concentrated with 0.45X AMPure PB beads for 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 the UC Davis DNA Technologies Core (Davis, CA) using six 8M SMRT cells (Pacific Biosciences, Menlo Park, CA; Cat #101-389-001) on the PacBio Sequel II with sequencing chemistry 2.0 and 30-hour movies.
SUB15451867
HMMgDNA0052