Lysis buffer

High molecular weight DNA was extracted both from fresh (<5 days old) and frozen (– 80 °C) whole blood. DNA extraction was performed following the manufacturer’s guidelines (PlugLysis, Bionano Genomics, USA). RBC lysis solution (Qiagen) was used to lyse red blood cells and pellet white blood cells. The white blood cells were re-suspended in cell suspension buffer (Bio-Rad) and embedded into agarose plugs (CHEF Genomic DNA Plug Kit, Bio-Rad) to lessen fragmentation of long DNA molecules during the overnight lysis at 50 °C using a 16:1 ratio of lysis buffer (Bionano Genomics, USA) and Puregene Proteinase K (Qiagen). The plugs were washed with Tris-EDTA buffer and digested at 43 °C with GELase (Epicentre). Extracted high molecular weight DNA was purified from digested materials/enzymes via drop dialysis using Millipore membrane filters (EMD Millipore, USA) placed on Tris-EDTA buffer. DNA quantifications were carried out using Qubit dsDNA assay kits with a Qubit 3.0 Fluorometer (ThermoFisher Scientific).

Whole leaves were sampled from S. bicolor accession Tx430 grown in the greenhouse for approximately 2 months. Samples were immediately collected in dry ice and stored at −80 °C until further handling. 25–30 g of frozen tissue was ground in liquid nitrogen prior to extraction of High Molecular Weight (HMW) genomic DNA from sorghum Tx430 adult leaf tissue using a protocol which was originally designed for the extraction of DNA for BAC library construction^{20 (link)}. For Bionano mapping, fresh tissue was collected from 3-week-old seedlings. HMW genomic DNA was prepared according to a protocol developed specifically by Bionano Genomics. Briefly, 2 g young leaf tissue was collected from live plants and immediately treated with a formaldehyde solution. After fixation, tissue was cut into approximately 2 × 2 mm squares and homogenized using a stator. Nuclei were isolated by gradient centrifugation and embedded into agarose plugs. After overnight proteinase K digestion in the presence of Lysis Buffer (Bionano Genomics) and 1 h treatment with RNase A, plugs were washed three times in 1× Wash Buffer (Bionano Genomics), then five times in 1× TE buffer. After the final wash, DNA was recovered by melting the plugs, digesting agarose with 2 µl of 0.5 U/µl Agarase, and dialyzing for 45 min in 1× TE buffer.