The leaf tissue from 3-week-old plants was collected and flash-frozen in liquid nitrogen. Genomic DNA was extracted from the leaf tissue using DNeasy Plant Maxi kit (Qiagen). For the Illumina PCR-free library, genomic DNA was fragmented in a Covaris S220 and separated on a SAGE-ELF (Sage Science) following the manufacturer’s instructions. The fraction that is 310~450 bp in size from SAGE-ELF were used for PCR-free library construction using TruSeq Nano DNA Library Preparation Kit (Illumina). The 20-kb PacBio library were prepared and sequenced on PacBio RS II using P6-C4 chemistry at Tianjin Biochip Corporation, following the manufacturer’s standard protocols.
Hi-C was performed following a published protocol48 (link). Briefly, 2 g of 10-day-old broomcorn millet seedlings were fixed in 1% formaldehyde solution. The nuclei/chromatin was extracted from the fixed tissue and digested with HindIII (New England Biolabs). The overhangs resulting from HindIII digestion were filled in by biotin-14-dCTP (Invitrogen) and the Klenow enzyme (NEB). After dilution and re-ligation with T4 DNA ligase (NEB), genomic DNA was extracted and sheared to a size of 300−500 bp with Bioruptor (Diagenode). The biotin-labeled DNA fragments were enriched using streptavidin beads (Invitrogen) and subject to library preparation.
Hi-C was performed following a published protocol48 (link). Briefly, 2 g of 10-day-old broomcorn millet seedlings were fixed in 1% formaldehyde solution. The nuclei/chromatin was extracted from the fixed tissue and digested with HindIII (New England Biolabs). The overhangs resulting from HindIII digestion were filled in by biotin-14-dCTP (Invitrogen) and the Klenow enzyme (NEB). After dilution and re-ligation with T4 DNA ligase (NEB), genomic DNA was extracted and sheared to a size of 300−500 bp with Bioruptor (Diagenode). The biotin-labeled DNA fragments were enriched using streptavidin beads (Invitrogen) and subject to library preparation.
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