RNA-seq of sugar beet: leaf, chilling at 4 degrees centigrade after 24 hours
The quality of total RNA was tested by Nanodrop and Agilent2100. Magnetic bead with Oligo(dT) was employed to enrich mRNA. Subsequently, NEB fragmentation buffer was added for breaking total RNA into fragments. The first chain of cDNA was reverse-transcripted by random hexamers, then dNTPs and DNA polymerase I were added for synthesis of the second chain. AMPure XP beads was used for purifying the double chain cDNA. Then, end repairing, A tail adding and adapter ligation were performed. After selection of fragment size by AMPure XP beads, USER was used for digesting cDNA containing U base. Finally, ssRNA-seq library was enriched by PCR.