Euplectellid hexactinellid (tulip sponge) PacBio HiFi sequecing, SMRT-cell 1
The sponge was sent to Brigham Young University DNA Sequencing Center, where they prepared one PacBio HiFi library. HMW DNA for the HiFi WGS library was extracted by first powderizing 500mg of sample under liquid nitrogen, then digesting the tissue in 5mL of 30mM Tris, 10mM EDTA, 1% SDS, 20L/mL proteinase K solution at pH 8.0 at 53C for 4 hours. The sample were spun down at 500 rcf at room temperature for 5 minutes to pellet debris. The supernatants were added to new tubes with 10mL of 96% EtOH, and then incubated at -20C for one hour. The cold mixtures were then centrifuged at 15k rcf at 4C for 10 minutes. The supernatants were removed, then washed with 80% EtOH. At this stage G2 buffer was added from the Qiagen Genomic-tip 20/G kit. The G2 solution and pellet mixtures were allowed to sit overnight in a refrigerator at 4C. These were then used in the Qiagen Genomic-tip 20/G protocol. The resulting HMW DNA samples was used in the PacBio Hifi library prep. The PacBio SMRTbell Express Template Prep Kit 2.0 + Enzyme Clean Up Kit 2.0 + Sequencing Primer v5 Bundle (PN: 102-088-900) was used to prepare the HiFi WGS library. The library of the hexactinellid sponge was sequenced in two Sequel II SMRT Cells (8M) on the 15-hour movie mode. CCS reads were called using the software bundled with PacBio SMRTtools release 10.1.0.119588. The 2 SMRT cells of the library from the hexactinellid sponge yielded 42.8 Gb of reads in 2,842,892 reads.