2500 ng of total RNA from human cells or 250 fmol of synthesized pre-miRNAs were ligated by T4 RNA ligase 1. The ligated pre-miRNAs were then reverse-transcribed by Superscripts IV (Invitrogen, 18090010) and RT primers that formed 25–30 base pairs with pre-miRNAs. The reverse complementary sequence of the 3'-adapter (5'- TGGAATTCTCGGGTGCCAAGG-3') was included in the RT primers. The resulting cDNAs were first amplified using the reverse primer (5'-CCTTGGCACCCGAGAATTCCA-3') and the forward primer containing the 5'-adapter sequence (5'- GTTCAGAGTTCTACAGTCCGACGATC-3'). The resulting PCR products were then amplified using RPIx and RP1 primers to generate the DNA libraries for next-generation sequencing (NGS). The DNA libraries were sequenced by Illumina Nextseq 500 sequencer using Nextseq Mid Output 1250 (2x75) cycles kit.