Nicotiana tabacum Chemical topping

Burley tobacco (Nicotiana tabacum variety 'KT210 LC') plants were grown at the Agricultural Experiment Station Spindletop Farm near Lexington, Kentucky, USA. Maleic hydrazide (MH, Royal MH-30, 180 g a.i. liter-1, United Agri Products, USA), was applied with a CO2-pressurized sprayer calibrated to 468 L ha--1 with a directed three-nozzle row-1 configuration (TG3 - TG5- TG3 coarse solid cone nozzles, TeeJet Inc., USA) at the 10% button stage. Axillary and apical bud samples were collected 24 h after MH application from the MH-treated and control plants (untreated), frozen immediately in liquid nitrogen and stored at _80 _C until RNA extraction (Fig. S1).Total RNA was isolated from 100 mg of ApB and AxB tissues using the RNeasy Plant Mini Kit (Qiagen, USA) following the manufacturer's instructions. Quality of the RNA samples was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and RNA samples with RNA integrity number (RIN) 8 or above were used for library preparation. Twelve cDNA libraries were prepared using the TruSeq RNA Sample Prep Kit (Illumina, USA) according to the manufacturer's protocol. Individually indexed samples were, then, were combined at equimolar proportions into three pools with 6 samples per pool and each pool was loaded onto a single lane of a flow cell. A 50 cycle single-end sequencing run was performed on the Illumina HiSeq2500 at Duke Center for Genomic and Computational Biology.