Hyb-seq of Cardamine rivularis Riv_ALE_5
Genomic DNA (400 ng per accession) was fragmented with a Covaris S220 or M220  sonicators (Woburn, MA, USA) to a target fragment size of 500 bp. Sequencing libraries were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina following the manufacturer's protocol (New England Biolabs, MA, USA). After cleanup of Adaptor-ligated DNA with QIAquick PCR Purification Kit (Qiagen), size selection was performed by SPRIselect beads (Beckman Coulter, MA, USA) to gain 500  600 bp long fragments. Amplification was performed with 8 cycles of PCR, using index primers from NEBNext Multiplex Oligos for Illumina. The amplified libraries were cleaned-up with AMPure XP beads (Beckman Coulter) and pooled equimolarly (24 accessions/pool). The pooled libraries were enriched by hybridization with the synthetized RNA baits (at 65C for 26 h) using the MYbaits kit, following the protocol v. 3.02 (Arbor Biosciences). The target-enriched libraries were PCR amplified for nine cycles with KAPA HiFi HotStart mix (Kapa Biosystems, Cape Town, South Africa), purified with QIAquick PCR Purification Kit, and submitted for sequencing with 150 bp paired end reads on an Illumina MiSeq system.