title: 2bRAD sequencing of attached and drifting forms of Furcellaria lumbricalis
design: DNA was extracted from Furcellaria lumbricalis using a modified version of a protocol for Fucus vesiculosus L. (see Panova et al. 2016 for unmodified protocol), where the CTAB buffer for Littorina saxatilis, which omits PVP-40, was used instead of the F. veciculosus CTAB buffer. This protocol removes phenolic compounds using acetone, contaminant metabolites using a CTAB extraction buffer with DIECA, polysaccharides using -mercaptoethanol and RNA using RNase A, after which the NucleoSpin  Plant II kit (Macherey-Nagel) is used, followed by DNA cleanup with the Zymo DNA Clean & Concentrator kit (Zymo Research). We deployed restriction-site associated DNA (RAD) 2b-RAD sequencing. This was done following an adapted protocol (by Wang et al. 2012; https://github.com/z0on/2bRAD_denovo). This implied digesting 50 ng of DNA per sample using restriction endonuclease enzyme BCGI (New England BioLabs), ligation of adaptors with partial overhang and barcodes, followed by amplification. Amplified fragments were run on a 2% agarose gel, from which they were extracted and cleaned using the NucleoSpin  Gel and PCR Clean-up kit (Machery-Nagel).