NsiI NEB#R0127

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	RADseq libraries were prepared from extracted DNA according to the method described by Etter et al [22, 23] using the enzymes SbfI-HF and NsiI (New England Biolabs), with 1μg of DNA starting material.

Protocol tips
RADseq libraries were prepared from extracted DNA according to the method described by Etter et al [22, 23] using the enzymes SbfI-HF and NsiI (New England Biolabs), with 1μg of DNA starting material.

Publication protocol

RADseq libraries were prepared from extracted DNA according to the method described by Etter et al [22, 23] using the enzymes SbfI-HF and NsiI (New England Biolabs), with 1μg of DNA starting material. The shearing step was performed on a Bioruptor+ sonication device (Diagenode) with 10 cycles of 30 seconds on, 1 minute off (high setting). Zymo DNA Clean and Concentrator kits (Zymo Research) were used for each clean-up step. The final PCR amplifications were run for 12 cycles. We did not pool samples after the P1 ligation — all samples were barcoded and processed separately until final quantification with a Qubit fluorometer (Thermo Scientific) before being combined proportionately for multiplexed sequencing. Four different barcodes were used for each sample to increase library complexity and assist in eliminating PCR errors in downstream processing.

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