PacBIo HiFi sequencing data for Melursus ursinus: male sample KB16889
DNA was extracted from the cultured cell lines from sloth bear, Melursus ursinus, male, ID=KB16889. After thawing, cells were centrifuged at 150 x g for 5 minutes and the supernatant was discarded. Cell pellet was lysed with 1 mL lysis buffer containing 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 25 mM EDTA, 0.5% (w/v) SDS and 100 g/ml Proteinase K overnight at room temperature. The lysate was treated with RNase A for 30 minutes at 37C and cleaned with equal volumes of phenol/chloroform using phase lock gels (Quantabio, Beverly, MA; 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 with 70% ethanol twice and resuspended in elution buffer (10mM Tris, pH 8.0). DNA concentration was measured by Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA). Total yield from KB16889 was 30 g. The purity of the DNA was accessed using a NanoDrop spectrophotometer. All the samples had 260/280 ratio of 1.9 and 260/230 ratio 2.0. The integrity of the high molecular weight (HMW) gDNA was verified on a Femto pulse system (Agilent Technologies, Santa Clara, CA) where majority of the DNA was observed in the fragments > 100 kilobases (kb).PacBio HiFi SMRTbell library for the sloth, sun and spectacled bear were constructed using the SMRTbell Express Template Prep Kit v2.0 (Pacific Biosciences [PacBio], Menlo Park, CA; Cat. #100-938-900) according to the manufacturer's instructions. High molecular weight (HMW) genomic DNA (gDNA) was sheared to a target DNA size distribution between 15  20 kb using Diagenodes Megaruptor 3 system (Diagenode, Belgium; cat. B06010003). The sheared gDNA was concentrated using 0.45X of AMPure PB beads (PacBio, Cat. #100-265-900). Then there was a 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 PippinHT system (Sage Science, Beverly, MA; Cat #HPE7510) to collect fragments greater than 8 kb. The 15  20 kb average HiFi SMRTbell library was sequenced at UC Davis DNA Technologies Core (Davis, CA) using three 8M SMRT cells for each bear species, Sequel II sequencing chemistry 2.0, and 30-hour movies each on a PacBio Sequel II sequencer.