The pretreated RNA was then fragmented by heating at 70°C for 3.5 min in fragmentation buffer (Ambion), followed by immediate chilling on ice and addition of 1 μl of Stop solution. Fragmented RNA was purified with the RNeasy MinElute kit (Qiagen) following the instructions of the manufacturer except 675 μL of 100% ethanol is used in step two, instead of 500 μl. Purified RNA was dephosphorylated in phosphatase buffer (New England Biolabs) with 5 U of Antarctic phosphatase (New England Biolabs) and 40 U of RNaseOut (Life Technologies) at 37°C for 30 min followed by 5 min at 65°C. After chilling on ice RNA was phosphorylated by addition of the following reagents; 5 μl of 10× PNK buffer, 20 U of T4 polynucleotide kinase (New England Biolabs), 5 μl of 10 mM ATP (Epicentre, Illumina), 40 U of RNaseOut, 17 μl of water. The reaction was incubated at 37°C for 60 min. Phosphorylated RNA was purified with the RNeasy MinElute kit (Qiagen) as described above. Purified RNA was concentrated to 6 μl by vacuum centrifugation on a SpeedVac (Eppendorf). One
