title: RNA-seq analysis of winter rye grains (cultivar Marusenka): milk stage
design: Rye kernels from cultivar Marusenka from the middle part of the spikes (1015 homogeneous spikes were used for each biological replicate)were fixed with liquid nitrogen and stored at -80C until RNA extraction. The collected samples were ground in a homogenizer (MM400). Total RNA from kernels was isolated using a CTAB extraction buffer (2% [w/w] acetyl trimethylammonium bromide, 1% [w/w] polyvinyl pyrrolidone, 0.1 mol/L Tris-HCl, 1.4 mol/L NaCl, 20 mmol/L EDTA [pH 8.0]). Further the buffer solution was mixed with a chloroform:isoamyl alcohol (24:1 [v/v]) solution in 1:1 (v/v) proportion. Following centrifugation, the upper phase was separated and mixed with both an RLT buffer (1/8 of the upper-phase volume) of the RNeasy Mini Kit (Qiagen) and a Plant RNA Isolation Aid (Ambion) (1/20 of the upper-phase volume). Further extraction followed the manufacturers instructions. The llibrary construction and sequencing were performed by the Novogene Corporation (China; https://en.novogene.com/). Raw reads from run were filtered to remove adapter sequences, contamination and low-quality reads with the BBDuk utility [BBTools, https://jgi.doe.gov/data-and-tools/bbtools/].