title: WGS of Rhinophis oxyrhynchus: historical specimen from natural history collection
design: We followed a modified version of the protocol reported by Meyer and Kircher (2010), without DNA fragmentation (Brace et al., 2018). For the blunt end repair step, a mix was prepared of buffer Tango (1x), dNTPs (100M each), ATP (1mM), T4 Polynucleotide Kinase (0.5U/L) and T4 DNA polymerase (0.1U/L, Thermo Fisher Scientific). 40L of this mix was added to 30L of DNA extract (total volume 70L), and incubated at 25C for 15 minutes and at 12C for 5 minutes. Samples were subsequently purified using a Qiaquick PCR Purification Kit (Qiagen). 200L of PB buffer was added to these samples, transferred to a spin column and centrifuged at 10,000 RPM for 1 minute. 650L PE buffer was then added into each column and spun, followed by a final step of dry spinning to discard any residual buffer. Each sample was eluted in 22L of EB buffer and then centrifuged. An adapter ligation mix was prepared by mixing T4 DNA Ligase Buffer (1x), PEG-4000 (5%), T4 DNA Ligase (0.125U/L, Thermo Fisher Scientific) and adapter mix (100M each). 20L of sample was added to 20L of this mix (total volume 40L), and incubated at 22C for 30 minutes, followed by the Qiaquick purification step as reported above. An adapter fill mix was prepared with ThermoPol Buffer (1x), dNTPs (each 25mM) and Bst DNA Polymerase Large Fragment (0.3U/L, Thermo Fisher Scientific). 20L of sample was added to 20L of master mix (total volume 40L), and incubated at 37C for 20 minutes, followed by the Qiaquick purification step as reported above. A mix was prepared of AmpliTaq Buffer (1x), MgCl2 (2.5mM), dNTPs (200M each), BSA (0.4mg/mL), AmpliTaq Gold (0.05U/L, Thermo Fisher Scientific), index primer P5 (0.4M), index primer P7 (0.4M). 40L of this mix was added to 10L of sample (total volume 50L). Three index PCR reactions were carried out per library, to avoid excessive volumes and in case one reaction failed. Cycling conditions were as follows:  94C(2), [20x 94C(40), 60C(40), 72C(40)], 72C(10). PCR was followed by a purification step in a regular post-PCR room with a Qiaquick PCR Purification Kit, making a final elution of 60L of library per sample.