GSM5068897: BCU001637_Early-Flowering; Crocus sativus; OTHER
Leaves were lyophilised and preserved at -80°C until analysis. DNA extractions were carried out using the commercial kit GeneElute Plant Genomic DNA Miniprep Kit (Sigma-Aldrich) according to the manufacturer's instructions with one minor modification. In detail, we added 4% w/w of PVP (polyvinylpyrrolidone; Soffritti et al., 2016) to each sample during DNA extraction to facilitate the removal of secondary metabolites, such as polyphenols, and to produce a purer DNA for the subsequent analysis. To carry out the methyl filtration enrichment of gene-related sequences, genomic DNA was digested using the restriction enzyme HpaII. This enzyme was selected because it is able to achieve very good gene space coverage and very good depletion of transposable elements, as also reported in Wischnitzki et al. (2015). In brief, the steps of the MRE-seq analysis are: 1) digestion of 300 ng of genomic DNA with the restriction enzyme HpaII and contemporary ligation of double strand adaptors to the generated fragments; 2) PCR amplification of the adaptor- ligated DNA; 3) removal of the adaptor sequences by PmeI digestion (the sequence of the adaptor includes the rare cutting site of the enzyme GTTTAAAC). This step is important to increase the length of the informative sequences; 4) sequencing of the generated DNA fragments was done on an Illumina MiSeq platform (300 bp paired-end reads). All steps were carried out exactly as reported in detail in Wischnitzki et al. (2015).