B73 naked DNA MNase-seq (intermediate)
For MNase digestion of naked DNA, we extracted DNA from a B73 developing ear using a DNeasy Plant Mini Kit (Qiagen) and digested the DNA with micrococcal nuclease (MNase; New England Biolabs, #M0247S) as follows: We prepared a master mix on ice with 140 ng/μl DNA, 82 gel units/μl MNase, 150 ng/μl BSA in 1X micrococcal nuclease buffer, then divided the master mix into five 150-μl aliquots, still on ice. We transferred all aliquots simultaneously to a 37°C water bath and removed one at a time at the following time points: 1 min, 2 min, 4 min, 6 min, 8 min, and 16 min (at which time the bulk of DNA had been digested to fragments of less than 200 bp in length). To stop the digestions, we immediately placed the samples on ice and added 150 μl of Histone Removal Buffer (1% SDS, 100 mM NaCl, 50 mM EDTA, 100 mM Tris-HCl pH 8) and 15 μl of 20 mg/ml Proteinase K (New England Biolabs, #P8102S). We incubated the samples for 45 minutes at 65°C to digest histones, then further purified the DNA by adding an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuging. We then added an equal volume (300 μl) of chloroform:isoamyl alcohol (24:1) to the supernatant and centrifuged again. We collected the second supernatant (240 μl) and precipitated the DNA at -20°C after adding 1 μl 20 mg/ml glycogen, 10 uL 5 M NaCl and 625 μl ethanol. We pelleted by centrifugation, washed with 75% ethanol, let it dry, and suspended in 100 μl 10 mM Tris pH 7.5. Finally, we size selected for ~150-bp fragments by gel electrophoresis (3.5% agarose), and extracted the DNA with a QIAquick gel extraction kit (Qiagen), and recovered ~100 ng of DNA. We selected the 1, 4, and 8-minute digestions for Illumina library preparation (the “mild”, “intermediate”, and “severe” digestions).    For all samples, we started with 100-300 ng of double-stranded, fragmented DNA and end-repaired and 5'-phosphorylated using the End Repair Module (New England Biolabs #E6050L), A-tailed with Klenow exo- (New England Biolabs #M0212S), ligated to adapters (8.3 nM) with a Quick Ligase Kit (New England Biolabs, #M2200S, and PCR amplified using Phusion 2X HF master mix (New England Biolabs #M0531S) in a 50-ul total volume containing 200 nM of each primer. Thermocycler conditions were as follows: An initial denaturation at 98°C for 30 seconds; no more than 12 cycles of 98°C for 30 seconds, 65°C for 30 seconds, and 72°C for 30 seconds; and a final extension at 72°C for 5 minutes. We purified DNA between each enzymatic treatment using Agencourt AMPure XP at a ratio of 1.8:1, except after PCR, where the ratio was 0.9:1. Prior to sequencing, we selected amplicons corresponding to ~150-bp inserts by gel electrophoresis on a 2.5% agarose gel and purified using a QIAquick gel extraction kit (Qiagen #28704). We used indexed PCR primers from the ScriptSeq Index PCR primers (Epicentre, #RSBC10948 and #SSIP1202) at a concentration of 200 nM. We used a Y adapter of the following two oligos at a concentration of 8.3 nM each: /5Phos/GATCGGAAGAGCACACGTCT and ACACTCTTTCCCTACACGACGCTCTTCCGATCT.
B73 naked DNA MNase-seq (intermediate)