Kronos_minus_SHAPE_replicate2
5 day old etiolated Kronos seedlings were harvested by carefully removing the remaining seed, and incubated with DMSO in incubation buffer at 22 degree for 15 minutes. The washed seedlings were then harvested and ground into fine powder in liquid nitrogen and applied to RNA extraction. PolyA-selected RNA was recovered and reverse transcribed. cDNA ligation was performed using Circligase ssDNA Ligase to ligate the 3 end of cDNA to a ssDNA linker for 12 hrs at 65 degree. Ligation product over 100 nt was recovered after gel separation. Purified ligated cDNA was amplified. The PCR products were purified using agarose gel to recover the fragments of 200-650 bp  and sequenced.