Hyb-Seq of Erysimum carniolicum Vojakc2
The Hyb-Seq libraries were prepared using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs, MA, USA) following the manufacturers protocol. Genomic DNA (250 ng per accession) was fragmented enzymatically, followed by end repair and dA-tailing with a single enzyme mix included in the kit. After the adaptor ligation, clean-up with the QIAquick PCR Purification Kit (Qiagen), size selection was performed by SPRIselect beads (Beckman Coulter, MA, United States) to produce 500600 bp long fragments. Amplification was performed with eight cycles of PCR, using index primers from NEBNext Multiplex Oligos for Illumina. The amplified libraries were purified with AMPure XP beads (Beckman Coulter) and pooled equimolarly (24 accessions/pool). The pooled libraries were enriched by hybridization with biotinylated Erysimum-specific RNA baits at 65 C for 26 h, using the myBaits kit and following the protocol v. 5.03 (Daicel Arbor Biosciences). The target-enriched libraries were PCR amplified for nine cycles using the KAPA HiFi HotStart mix (Kapa Biosystems, Cape Town, South Africa), purified with the QIAquick PCR Purification Kit, and pooled with an unenriched library fraction in a 2:1 ratio. After the final purification step with 0.9x AMPure XP beads, the libraries were sequenced with 150-bp paired-end reads on an Illumina MiSeq system at BIOCEV, Czechia.