title: Exome capture of barley blf13 WT pool
design: Preparation of exome libraries was done according to SeqCap EZ HyperCap workflow (Roche, Basel, Switzerland) using KAPA High Throughput Library Preparation Kit, SeqCap Adapter Kit A with TrueSeq Index Adapters and dedicated SeqCap EZ reagent Kits (Roche, Basel, Switzerland). Agilent Bioanalyzer DNA 1000 assays (Agilent Technologies, Santa Clara, CA, USA) were used to determine the distribution of fragments sizes of each library. Concentration and purity of the libraries were estimated using NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Final measurement of library concentration was done using qPCR analysis with KAPA Library Quantification Kit (Roche, Basel, Switzerland). The detailed protocol for library preparation was described in (Rotasperti et al., 2022). Briefly, 700 ng genomic DNA extracted from pools of F2 individuals with contrasting phenotypes from mapping population was subjected to restriction digestion using KAPA HyperPlus Library Preparation Kit. Restriction was followed by end-repair and A-tailing, ligation of adapters with specific index sequences, dual-size selection and ligation-mediated-PCR (LM-PCR) steps, which were conducted according to manufacturers protocol (Roche, Basel, Switzerland). Dual-size selection narrowed down fragment sizes to between 150 - 500 bp, with the main peak at 310 bp. After LM-PCR the fragmented libraries were hybridized to the exome capture probes. The probes followed the design of 120426_Barley_BEC_D04.EZ, as described by Mascher et al., (2013) and cover 60 Mbp of barley exome, representing approximately 40,000 genes (Bayer et al., 2019). The hybridization step was performed at 47C for 18 h and was followed by series of washes with buffers of decreasing stringency. The recovery of captured library, post-capture PCR and final purification of the libraries was performed using AMPure beads (Beckman Coulter, Indianapolis, IN, USA).