Sequencing Targeted capture of Quercus robur and Quercus petraea : buds
Custom targeted genome bisulphite sequencing was performed with SureSelect  XT Methyl-Seq Target Enrichment (Agilent, Santa Clara, CA, USA) according to the manufacturers recommendations. We assessed the impact of five experimental variables probe dilution (1:1, 1:8, 1:10 and 1:16), amount of input DNA (500 ng, 600 ng, 750 ng, 935 ng, 1000 ng and 3000 ng), DNA fragmentation technique (acoustic vs. enzymatic shearing) and number of PCR cycles (14 or 15 cycles). The libraries were fragmented into fragments of about 200 base pairs, which were then subjected to end repair, A-tailing and the ligation of methylated adaptors before hybridisation with custom probes for 16 h. Bisulphite conversion was then performed with the EZ-DNA Methylation-Gold Kit (Zymo Research, Irvine, CA). A first PCR was performed with eight or nine cycles, followed by a 2 nd PCR with six cycles for indexing. Final libraries were quantified by fluorescence with the Quant-iT dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific) and pooled to an equimolar concentration. The size of the pool was assessed on a TapeStation 4200 system (Agilent Technologies) and its concentration was estimated by qPCR on a LC480 II system (Roche, Basel, Switzerland) using the QIAseq Library Quant kit (Qiagen, Hilden, Germany).