DNase-seq

For each inner stem tissue sample (V2 stage) and inner husk leaf sample, nuclei were extracted from 12 V2 stage maize seedlings and three husks according to the protocol of Steinmüller and Appel [98]. For each tissue, two biological replicate samples were used. Briefly, tissue was ground in liquid nitrogen, 5 g were transferred into an ice-cold 50 mL centrifuge tube, 25 mL of cold nuclei isolation buffer (20 mM Tris-HCl pH8, 250 mM sucrose, 5 mM MgCl2, 5 mM KCl, 40% glycerol, 0.25% Triton X-100, 0.5 mM EGTA pH 8, 5 mM EDTA pH8, 0.1 mM PMSF, 0.1% 2-mercaptoethanol, 1:1000 Proteinase Inhibitor Cocktail (Sigma)) were added and the tube was flicked until the powder was in suspension. The tube was rotated at low speed at 4 °C until the sample was completely thawed (about 30 min). The tissue suspension was filtered through successive layers of 60 μm and 20 μm nylon mesh (Nylon Net Filters, Millipore) into an ice-cold 50 mL centrifugation tube and centrifuged at 6000 × g for 15 min at 4 °C. The supernatant was discarded and the pellet resuspended in 15 mL of ice-cold nuclei isolation buffer using a 1 mL cutoff pipette tip, followed by centrifugation at 6000 × g for 12 min at 4 °C. The pellet was resuspended in 10 mL of ice-cold nuclei isolation buffer and centrifuged at the same conditions again, followed by resuspending the pellet in 1 mL of ice-cold nuclei storage buffer (20% glycerol, 20 mM Tris pH 7.5, 5 mM MgCl2, 1 mM DTT). To check the quality and abundance of the nuclei, a 20-μL aliquot was stained with 1 μL DAPI (1 mg/mL) and examined by fluorescent microscopy. The nuclei suspensions were flash frozen in liquid nitrogen and stored at –80 °C until further use.

DNase I treatment was adapted from Chandler et al. [99]. Nuclei suspensions were thawed on ice while preparing the solutions for DNase I digestion. One undigested control and four concentrations of DNase I (50, 100, 150 and 200 U/mL) were used (Additional file 1: Figure S15). In total, 2.5 mL of DNase I buffer (50 mM Tris pH8, 250 mM sucrose, 100 mM KCl, 0.1 mM CaCl2, 5 mM MgCl2, 50 μg/mL BSA, 0.05 M beta mercaptoethanol) was prepared per sample. The DNase I dilutions were prepared by mixing DNase I (Roche) with DNase I dilution buffer (20 mM Tris pH7.5, 50 mM NaCl, 1 mM DTT, 100 μg/mL BSA, 50% glycerol). A total of 1 mL of nuclei suspension was divided in 5 × 200 μL in 1.5-mL microcentrifuge tubes using cutoff pipette tips. The tubes were centrifuged at 1500 × g for 5 min at 4 °C and the supernatant was discarded. A total of 100 μL of 100 mM EDTA pH 8, followed by 600 μL of phenol/chloroform/isoamylalcohol (25:24:1 v/v), were added to the tube for the undigested control and set aside at room temperature after thorough mixing. The other pellets were resuspended in 475 μL of cold DNase I buffer by rubbing the tubes against a plastic tube rack and letting them incubate for 3 min at 25 °C. In total, 25 μL of each of the DNase I dilutions were added to the respective tubes with nuclei suspensions and incubated for 10 min at 25 °C. The reaction was stopped by adding 100 μL of 100 mM EDTA pH 8, mixing and adding 600 μL of phenol/chloroform/isoamyalcohol. All samples, including the undigested control, were shaken by hand or using a tissue lyser (Qiagen) at 8 Hz for 5 min. A second phenol/chloroform/isoamyalcohol extraction was performed, followed by an RNase A treatment (2 μg/mL final concentration) at 37 °C for 10 min. Totals of 600 μL isopropanol, 50 μL of 7.5 M ammonium acetate and 2 μL of 10 mg/mL glycogen were added followed by centrifugation at 16,000 × g for 30 min at 4 °C. Two 70% ethanol washings were performed and the pellets were finally resuspended in 30 μL 10 mM Tris-HCl pH 8.5. The concentration of nuclei acids was then assessed spectrophotometrically (Nanodrop, ThermoScientific) and the entire sample (30 μL) was mixed with 6 μL Cresol Red loading buffer (1.75 M sucrose (60%), 5 mM cresol red, pH 8) and loaded on an agarose gel (1× TAE buffer, 1.5% agarose, 0.5 μg/mL ethidium bromide). Gel visualisation under ultraviolet light indicated which digestion fulfilled the requirement that the DNA is only partially digested (Additional file 1: Figure S15). In our hands, these were the samples digested with 50 U/mL of DNase I. One should test several concentrations as the digestion efficiency can vary depending on the batch of DNase I enzyme and chromatin concentration. The DNA fractions in the range of 100–300 bp were extracted from the gel using gel purification (NucleoSpin Gel, Macherey Nagel) and the DNA was eluted from the column in 15 μL of 10 mM Tris-HCl pH 8.5. The DNA concentration was measured using Quant-iT PicoGreen (Invitrogen) on a fluorometer (Synergy 4 Hybrid Multi-Mode Microplate Reader, BioTek). A DNA concentration range of 1–3 ng/μL was obtained.

gDNA was extracted from 100 mg of inner husk tissue derived from three pooled husks using the DNeasy Plant Mini kit (Qiagen) and following the manufacturer’s instructions. A total of 1.7 μg of gDNA was digested with 50 U/mL of DNase I following the same protocol as described for chromatin.

DNA samples were diluted to 1 ng/μL in a total volume of 10 μL followed by library preparation using the Ovation Ultralow DR Multiplex kit (NuGEN) according to the manufacturer’s protocol. Fifteen cycles of amplification were performed for the naked DNA sample and 16–18 cycles for the chromatin-derived samples. The libraries were sequenced on an Illumina Hi-Seq2500 platform and approximately 20–30 million 100-bp single-end reads were obtained for each library.