title: double-digest RADSeq of Theromaster
design: We used restriction digest enzymes EcoRI-HF and Msp1 (New England Biolabs, Ipswich, MA), and the corresponding adapters from Peterson et al. (2012). Briefly, ~500 ng of genomic DNA was digested for 3 hours in a 50 l reaction with 100 units each of the restriction enzymes EcoRI-HF and Msp1 (New England Biolabs, Ipswich, MA), and 1X CutSmart Buffer (New England Biolabs). Samples were purified using Agencourt AMPure XP bead cleanup (Beckman Coulter, Inc., Brea, CA). Adapters were ligated to digested DNA in a 40 l reaction that consisted of 33 l digested DNA, 1.05 M MspI P2 adapter, 0.54 M EcoRI P1, 400 units of T4 DNA-ligase, and 1X T4 DNA ligase reaction buffer (New England Biolabs). Ligation reactions were incubated at room temperature for 40 minutes, heat killed at 65C for 10 minutes, then cooled to room temperature at a rate of 2C per 90 seconds. Samples with different adapters were pooled by column and then purified using AMPure XP bead cleanup. Pooled samples were then size selected to a size range of 400600 bp with a Pippin Prep automated size-selection instrument (Sage Science, Beverly, MA). Primers with Illumina indices were added to the pooled samples using PCR; 50 l reactions consisted of 23 l DNA template, 2 uM PCR Primer P1, 2 uM PCR primer P2 (eight types for second-tier multiplexing, one per pooled sample), and 1X Phusion High Fidelity PCR Mastermix (New England Biolabs). Cycle conditions were 98C for 30 seconds, 12 iterations of 98C for 10 seconds and 72C for 20 seconds (with a 16% ramp to slow cooling), and 72C for 10 minutes. PCR products were purified via AMPure XP bead cleanup and quantified using a Bioanalyzer (Agilent Technologies, Santa Clara, CA). A pool consisting of an equimolar quantity of each library was sequenced as 100 bp SE reads on the Illumina HiSeq2500 platform at the University of California, Riversides Institute for Integrative Genomics Biology  Genomics Core Facility.