Alternative fragmentation methods. (a) RNaseIII was used for the standard fragmentation method. (b) Heat fragmentation method was used as an alternative fragmentation method. After heat fragmentation, T4PNK was applied to modify RNA fragment ends for adaptor ligation. T4PNK adds phosphate and leaves hydroxyl groups at their 5’ and 3’ ends, respectively. (c) Modified heat fragmentation method was designed using OptiKinase. OptiKinase reduces T4PNK bias introduced during the RNA fragment modification. Because OptiKinase has only kinase activity, T4PNK was used for phosphatase activity after OptiKinase treatment.