ezRAD of Antipatharia
We generated ezRAD reduced-representation genomic libraries (Toonen et al., 2013) following the protocol of Knapp et al. (2016). Briefly, we digested genomic DNA with the isoschizomers MboI and Sau3AI (New England Biolabs), which recognize 5 GATC cut sites. We digested a total of 375 ng of genomic DNA from each sample using 2 l of each MboI and Sau3AI in a 50 l reaction volume following manufacturer protocols. We incubated each digestion in a thermocycler at 37C for 3 hours, followed by inactivation at 65C for 20 minutes. Digestion was verified visually by running each digest beside undigested genomic DNA on a 1% agarose gel. DNA fragments were size-selected (300-600 bp) using a Pippen Prep (Sage Science, Beverly, MA, USA) before preparing genomic libraries for sequencing using either the Illumina TruSeq Nano (Illumina, San Diego, CA, USA) or Watchmaker DNA Library Preparation (Watchmaker Genomics, Boulder, CO, USA) kits. Following bioanalyzer and qPCR control checks, libraries were sequenced on the Illumina MiSeq (V3 2x300 bp paired-end) at the IIGB Genomics Core facility at UC Riverside or the Advanced Studies in Genomics, Proteomics and Bioinformatics (ASGPB) at the University of Hawaii.