title: PacBio Sequencing of Dissostichus eleginoides genome: Flowcell 2
design: High molecular weight DNA was extracted from material stored in RNAlater (ThermoFisher) using the Qiagen Genomic tip 500G and the manufacturers recommended protocol. Briefly, 400 mg of toothfish visceral tissue (spleen, kidney and liver) was ground to a powder with liquid nitrogen in a pre-cooled mortar and pestle and then digested in 20 ml of G2 buffer containing RNAse and proteinase K for 2 h at 50C. Following digestion, the sample was loaded onto a pre equilibrated genomic tip 500G. The column was washed twice with 15ml Buffer QC and eluted 15 ml buffer QF. The DNA was precipitated using 0.7 volumes (10.5 ml) isopropanol and centrifugation at 5000g for 15min at 4C; the pellet was washed with 4 ml cold 70% ethanol and centrifuged at 5000g for a further 10 min at 4C. The DNA pellet was air dried for 10 min and resuspended in 500 l TE buffer and stored at -80C.Genomic DNA libraries were generated using the SMRTbell Express Template Prep Kit 2.0, following the protocol described in ref 1. An additional enzymatic digest step (to remove any linear molecules) was included as described on page 13 of ref 2, before size-selection of >20Kb on a high pass cassette on the Blue Pippin (Sage Science, MA, USA). A total of 6 SMRT cells were run on the PacBio Sequel. Ref1:https://www.pacb.com/wp-content/uploads/Procedure-Checklist-Preparing-gDNA-Libraries-Using-the-SMRTbell-Express-Template-Preparation-Kit-2.0.pdfRef2:https://www.pacb.com/wp-content/uploads/Procedure-Checklist-Preparing-HiFi-Libraries-from-Low-DNA-Input-Using-SMRTbell-Express-Template-Prep-Kit-2.0.pdf