GSM5289576: Product, biological replicate 4, technical replicate 2; Homo sapiens; miRNA-Seq
RNAs were obtained by phenol extraction and ethanol precipitation. The eluate was incubated with 0.5 μl of 10 μM 3′ adapter (Illumina, TruSeq Small RNA Library Prep Kit), which is modified with additional six degenerated bases at the 5′ end, at 70°C for 2 min. The 3′ adapter-ligated fragments were dissolved in 3 μl of TDW and incubated with 0.5 μl of 10 μM 5′ adapter (Illumina, TruSeq Small RNA Library Prep Kit), which is modified with additional six degenerated bases at the 3′ end, at 70°C for 2 min. The adapter-ligated fragments were reverse-transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) and RT primer (Illumina, TruSeq Small RNA Library Prep Kit). For Illumina sequencing, cDNA was amplified by PCR that utilized Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) and PCR primers from TruSeq Small RNA Library Prep Kit (Illumina). Meanwhile, input substrates were also reverse-transcribed using custom RT primers and PCR-amplified using PCR primers from TruSeq Small RNA Library Prep Kit (Illumina).