Genomic tip columns 500 g

Allodiplogaster sudhausi nematodes were washed off of 100 NGM agar plates using M9 buffer and pelleted by centrifugation at 1,300 × g for 1 min. The pellet was washed twice in M9 before worms were frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. The powder was directly transferred into the lysis buffer from the QIAGEN genomic DNA extraction kit, which was used in combination with QIAGEN genomic tip columns (500/G) (QIAGEN, Hamburg, Germany). The protocol was performed following the manufacturer's instructions. All steps involving vortexing of the sample were replaced by inversion to limit unwanted DNA shearing. DNA quality and quantity were determined with a NanoDrop ND 1000 spectrometer (PeqLab, Erlangen, Germany), a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, USA), and by a Femto pulse system (Agilent, CA, USA). A total of 20 μg A. sudhausi genomic DNA was sheared to a target fragment size of 45 kb using a needle. A 45-kb template library was prepared using the BluePippin size-selection system according to the manufacturer's protocol (P/N 100-286-000-07, Pacific Biosciences, California, USA). The final library was sequenced on a Pacific Biosciences Sequel instrument following the Magbead loading protocol and version 1.2.1 sequencing kits. A total of two SMRT cells (version 1.2.1) generated 40 Gb (100-fold coverage).