RNA-seq of the Non-cold tolerant cultivar under cold treatment
Seeds of Ningjiang3 (the non-cold tolerant cultivar) were sowed in pots in a incubator set to 25℃ and 14h light per day. When treatment started, half of the two-week-old seedlings were transferred to another incubator set to 4℃, other conditions remain unchanged.24 hours later, one well developed leaf was harvested from each seedling.More than 5 seedlings were collected and pooled for each cultivar. These collected leaves were frozen in liquid nitrogen immediately and stored at −80 °C.  Total RNA was extracted from the collected leaves using RNAiso Plus (TaKaRa Biotechnology CO., LTD). The quality of RNA was determined by a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc. Waltham, MA, USA). RNA integrity was confirmed by electrophoresis on 1.5 % agarose gel. Approximately 20 μg of total RNA was used for Illumina sequencing at Biomarker technologies (Beijing, China). cDNA library construction was performed via following steps. First, mRNA was purified with biotin-Oligo (dT) magnetic bead and was randomly sheared by Fragmentation Buffer. Then using mRNA as template, First strand cDNA was synthesized with random hexamers, then buffer、dNTPs、RNase H and DNA polymerase I were added to synthesis second-strand cDNA, and the product was purified with AMPure XP beads. Afterwards, purified double-strand cDNA was end-repaired, poly-A tail-added and ligated to Illumina adapter, AMPure XP beads were used to select fragment size. Finally, cDNA library was obtained by PCR enrichment. Sequencing of the purified library was carried out on an Hiseq2500 (Illumina Inc., USA).