title: RADseq of Cardamine acris subsp. acris 260BAB_102
design: Double-digest RADseq libraries were prepared according to the protocol of Peterson et al. (2002) with some modifications. Genomic DNA was purified with 0.4x AMPure XP beads (Beckman Coulter), diluted to 800 ng and digested at 37 C for 2 hours with 4 units of HpyCH4V in CutSmart buffer in a total volume of 48 uL, followed by an inactivation step at 65 C for 20 minutes. The digested DNA was purified with 1.5x AMPure XP beads, diluted to 100 ng and subjected to A-Taling with 15 units of Klenow fragment (New England Biolabs), 0.04 mM dATP, 1x NEB2 buffer in a volume of 30 uL at 37 C for 30 minutes, followed by ligation of adapters (0.08 uM) with 600 units of T4 DNA ligase, 1 mM ATP (New England Biolabs) and 2.5 mM MgCl2 in a total volume of 40 uL. Adaptor ligation was performed at 23 C  for 30 minutes, heat-killed at 65 C for 10 minutes and cooled slowly to room temperature (cooling at approximately 2 C every 90 seconds). The P5 and P7 adapters had the same sequences and contained a 5-base pair-long barcode. The samples barcoded with different adapters were pooled and then purified with 1.2x AMPure XP beads. The pooled ligation products were size-selected with Pippin Prep (Sage Science, Inc., MA, USA) using 2% agarose gel cassettes, with the size range set to 302-398 bp. PCR amplification was then performed using a Phusion HiFi (New England Biolabs) polymerase with 10 cycles. The PCR mix contained 5 uL of the size-selected ligation product, 0.4 units of Phusion HiFi, 1x Phusion HF buffer, 0.2 mM of each dNTPs, 2 uM of the universal P5 primer, 2 uM of the indexed P7 primer (with different 6-base pair-long indexes used for each pool) and completed with water to a total volume of 20 uL. The amplified libraries were purified with 1.5x AMPure XP beads, quantified using the Qubit 2.0 fluorometer (ThermoFisher Scientific, MA, USA), and the libraries containing different P7 primers were pooled equimolarly to form a final ddRADseq library. The library was submitted for sequencing with 150-bp paired end reads on Illumina systems (HiSeq, NextSeq, or NovaSeq) to EMBLEM Technology Transfer, Heidelberg, Germany, or to Novogene, Cambridge, UK.