title: PacBio HiFi reads for Phoronis australis
design: High-molecular-weight DNA was extracted from the ampulla of a single adult P. australis individual using chaotropic lysis buffer (CHAOS buffer: 4 M guanidine thiocyanate, 0.5% N-lauroyl sarcosine sodium, 25 mM Tris-HCl pH 8.0, and 0.1 M 2-mercaptoethanol), followed by phenol/chloroform/isoamyl alcohol (PCI; 25:24:1) extraction. In brief, the tissue was homogenized in 250 L of CHAOS buffer and incubated overnight at 55C. The lysate was mixed with 250 L of DNA extraction buffer (100 mM Tris-Cl pH 8.0, 10 mM EDTA, 0.1% SDS), vortexed with 250 L of PCI solution, and transferred to a pre-spun Phasemaker Tube. The mixture was centrifuged at 15,000  g for 5 min at 4C, and the aqueous phase was transferred to a new tube. DNA was precipitated by adding 600 L of isopropanol, followed by gentle mixing and centrifugation at 15,000  g for 20 min at 4C. The pellet was washed twice with 1 mL of 70% ethanol, each wash followed by centrifugation at 15,000  g for 10 min at 4C. After air-drying for 5 min, the DNA pellet was dissolved in UltraPure DNase/RNase-Free distilled water. Genomic DNA was separately extracted from three phoronid individuals and quantified using a NanoDrop One spectrophotometer and a Qubit 4 fluorometer. DNA size and integrity were assessed with the Agilent Femto Pulse system, and the genomic DNA Quality Number (GQN) was calculated using a 10 kb threshold. The sample with the highest GQN (8.3) and a size peak of 113.7 kb was selected for library preparation. The selected gDNA was sheared using a Megaruptor 2 and size-selected with a BluePippin system to isolate fragments longer than 10 kb. A whole-genome library was prepared using the SMRTbell Prep Kit 3.0, resulting in a library with a peak size of 19 kb. The library was sequenced on a PacBio Sequel IIe platform using one SMRT Cell, producing 6.2 million reads, including 1.8 million HiFi reads, totalling 33.4 Gb with a mean read length of 18 kb.