RADseq of pomatoschistus flavescens: individual 109
DNA was extracted from fish caudal muscle using the E-Z 96 Tissue DNA kit (Omega Bio-tek) and treated with RNAse (#19101). Concentrations were measured with a Qubit instrument (Thermo Fisher Scientific) and normalized to 7 ng/l. The double-digest Restriction Associated DNA (ddRAD) protocol was initiated by fragmenting the normalised DNA with two restriction enzymes, PstI and MseI. Fragments were ligated with paired-end oligo adapters that include variable length barcodes (5-7bp), before pooling of individual samples into libraries. Size selection with the BluePippin instrument (SageScience) was conducted to ensure DNA fragment sizes in the range 250-600 bp. To the libraries were added 5% PhiX prior to 150 bp paired-end sequencing on a HiSeq instrument (Illumina). Three technical replicates were made per individual, totalling to an average of 7 million reads per individual.
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109