Broadcasters Montastrea cavernosa and Siderastrea siderea sampled in the Lower Florida Keys and brooders Agaricia agaricites and Porites astreoides sampled in Florida coral reefs, from Dry Tortugas to Fowley Rocks. The first broadcasted transect was sampled in 2015, the second broadcaster transect sampled in 2017. Brooders were sampled in 2019

Tissue samples were collected using hammer and chisel and kept on 100% ethanol and the minimum logistically feasible temperature (-20C or -80C) until processed.

Genomic DNA was extracted from tissue samples using a modified phenol-chloroform procedure. First, samples were transferred to an extraction solution comprising 800 μL CTAB extraction buffer (2% CTAB, 100 mM Tris pH 8, 20 mM EDTA, 1.4 M NaCl ), 1.6µL beta merceptoethanol, 1µL proteinase K, and 1µL RNAse A. Tissue was separated from the skeletal matrix and macerated by bead beating in a BioSpec MiniBeadbeater-96 for 15 seconds using 150-212 μm glass beads and then incubated at 42°C for at least one hour. Skeletal fragments were pelleted by spinning in a tabletop centrifuge at maximum speed for 15 minutes. The aqueous phase was then transferred to a clean tube, and 800 µL phenol-chloroform/isoamyl alcohol was added and vortexed into solution for 2-3 seconds. Samples were centrifuged at maximum speed at 4°C for 20 minutes to separate the DNA from organic contaminants. The aqueous supernatant was transferred to a clean tube to which 550 µL ice cold isopropanol was added and gently mixed by inverting. After incubating at -20°C for 20 minutes, samples were again centrifuged at 4°C and maximum speed for 20 minutes to pellet DNA. The supernatant was discarded, 1 mL 80% ethanol added, and the sample was centrifuged at 4°C and maximum speed for 5 minutes. The supernatant was discarded and sample tubes were allowed to air dry for 15-20 minutes. Lastly, pelleted DNA was dissolved in 30 µL warm (65°C) nuclease-free water. Isolated DNA was then further purified using the Zymo Clean and Concentrator-10 kit and normalized to a concentration of 12.5 ng/μL.

2b-RAD libraries were prepared according to the protocol maintained at https://github.com/z0on/2bRAD_denovo (see related software publications). Briefly, 50 ng DNA was then digested in a reaction comprising 1 U BcgI restriction enzyme, 1X NEB Buffer #3, 20 μM SAM in a total reaction volume of 6 μL. Digests were incubated at 37°C for one hour followed by 20 minutes at 65°C to inactivate the enzyme. Barcodes were then attached in two stages, first by ligation to the digested DNA fragments and secondly via PCR amplification. Ligation reactions consisted of 1X T4 ligase buffer, 400 U T4 ligase, and 0.25 μM of two double-stranded ligation adaptors, one of which contained an internal barcode, combined with 6 μL digested DNA in a total reaction volume of 20 μL. Ligation reactions were held at 4°C for 12 hours followed by 20 minutes at 65°C to inactivate the enzyme. Samples with unique ligation barcodes were then pooled and a second barcode was attached via PCR with thermocycler conditions set to 30 seconds at 70°C, followed by 15 cycles of 20 seconds at 95°C, 3 minutes at 65°C and 30 seconds at 72°C. PCR was carried out using 10 μL of pooled ligations with 1X Titanium Taq polymerase, 1X Titanium Taq buffer, 200 μM dNTPs, 0.12 μM unique ILL-BC barcode, 0.12 μM TruSeq-UN barcode, and 0.2 μM each of P5 and P7 adaptors in a total reaction volume of 50 μL.

RNA for TagSeq was isolated using RNAqueous kit (Ambion). DNA contamination was removed by treatign with RNA-seq free DNAse (Ambion). The TagSeq libraries were prepared following the “simplified” version of the protocol, which is maintaned on GitHub (see related software publications).