NextSeq 500 paired end sequencing; ATAC-seq of the B104 genome and the B73 control
ATAC-seq of the B104 genome and the B73 control
1st and 2nd leaves and primary root were harvested 6 days after planting. Soil was saturated with tap water and placed under a 50/50 mixture of 4,100 K (Sylvania Supersaver Cool White Delux F34CWX/SS, 34 W) and 3,000 K (GE Ecolux with starcoat, F40CX30ECO, 40 W) light. The photoperiod was 16 h of light and 8 h of dark. The temperature was approximately 25 °C during light hours. The relative humidity was approximately 54%. Harvested tissue was placed in 2 ml of pre-chilled lysis buffer (15 mM Tris–HCl pH 7.5, 20 mM sodium chloride, 80 mM potassium chloride, 0.5 mM spermine, 5 mM 2-mercaptoethanol and 0.2% TritonX-100). The chopped slurry was filtered twice through miracloth and once through a 40-μm cell strainer. The crude nuclei were stained with 4,6-diamidino-2-phenylindole and loaded into a flow cytometer (Beckman Coulter MoFlo XDP). Nuclei were purified by flow sorting and washed in accordance with Lu et al (doi: 10.1093/nar/gkw1179). The sorted nuclei (50,000 nuclei per reaction) were incubated with 2 μl of transposome in 40 μl of tagmentation buffer (10 mM TAPS–sodium hydroxide pH 8.0, 5 mM magnesium chloride) at 37 °C for 30 min without rotation. The integration products were purified using a Qiagen MinElute PCR Purification Kit and then amplified using Phusion DNA polymerase for 11 cycles. PCR cycle number was determined as described previously (10.1038/nmeth.2688). Amplified libraries were purified with AMPure beads to remove primers. To make the ATAC-seq control, nuclei were sorted and genomic DNA was extracted from maize leaves using the Qiagen DNeasy Plant Mini Kit (cat. no. 69106). Then ~1 ng of gDNA was incubated with 2 μl of transposomes in 40 μl of tagmentation buffer at 37 °C for 30 min. All procedures after this were identical to the standard ATAC-seq library protocol described here.