title: Hyb-seq of Cardamine lazica lazTREU4
design: Sequencing libraries were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, MA, USA) following the recommended manufacturers protocol. Genomic DNA (400 ng per accession) was fragmented with Covaris M220 sonicator (Woburn, MA, USA) to a target fragment size of 500 bp. Adaptor-ligated DNA fragments were purified with the QIAquick PCR Purification Kit (Qiagen) and size-selected using SPRIselect beads (Beckman Coulter, MA, USA) to 500600 bp size range. PCR enrichment with eight cycles was performed using index primers from NEBNext Multiplex Oligos for Illumina. The amplified libraries were cleaned up with AMPure XP beads (Beckman Coulter), measured with Qubit 2.0 fluorometer (ThermoFisher Scientific), and pooled equimolarly (24 accessions/pool). The pooled library was size-selected using SPRIselect beads as above and measured again with Qubit 2.0. An aliquot containing 250 ng was enriched by hybridization with synthesized RNA baits (26 h at 65C) using the MYbaits kit, following the protocol v. 3.02 (Arbor Biosciences). The target-enriched library was amplified by PCR with nine cycles using the KAPA HiFi HotStart mix (Kapa Biosystems, Cape Town, South Africa) and purified with the QIAquick PCR Purification Kit. Enriched and unenriched library aliquots were pooled in a ratio 2:1, finally purified with AMPure XP beads, and submitted for sequencing with 150 bp paired end reads on an Illumina MiSeq system.