RNeasy Mini Kit; Qiagen Messenger RNA was purified from total RNA using poly-T oligo-attached  magnetic beads. After fragmentation, the first strand cDNA was synthesized  using random hexamer primers, followed by the second strand cDNA  synthesis using either dUTP for directional library or dTTP for non-directional  library.For the non-directional library, it was ready after end repair, A-tailing,  adapter ligation, size selection, amplification, and purification.For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, andpurification.