DNA Digestion and Library Preparation

The libraries were prepared by following the protocol developed by Qi et al. [60 (link)]. Briefly, genomic DNA was digested using MspI and PstI-HF (NEB) at 37℃ for 2 h, then, to inactivate the restriction enzymes at 75℃ for 20 min. The barcoded PstI-HF adapter and common MspI adapater were respectively ligated on the corresponding restriction sites of all samples by T4 DNA ligase (NEB). Ligation reaction lasted for 2 h at 22℃. Following ligation, fragments less than 300 bp were filtered out. PCR amplification was done for each sample separately. PCR products were checked on a 1.0% agarose gel. Primers, dNTP and small DNA fragments were removed from the pooled DNA. Final libraries were sequenced using Illumina Hiseq Xten, PE150 Platform.