GSM5931247: miD0_2; Mus musculus; miRNA-Seq
Total RNA was extracted according to the manufacturer's instructions using TRIzol reagent (Invitrogen, USA). RNA integrity was assessed using an Agilent Bioanalyzer 2100 system (Agilent Technologies). A total amount of 3μg total RNA per sample was used as input material for the small RNA library. Briefly, NEB 3' SR Adaptor was directly, and specifically ligated to 3' end of miRNA, siRNA, and piRNA. After the 3' ligation reaction, the SR RT Primer hybridized to the excess of 3' SR Adaptor (that remained free after the 3' ligation reaction) and transformed the single-stranded deoxyribonucleic acid (DNA) adaptor into a double-stranded DNA molecule. 5´ends adapter was ligated to 5´ends of miRNAs, siRNA, and piRNA. After synthesis of the first-strand cDNA, PCR amplification is performed. DNA fragments corresponding to 140~160 bp (the length of small noncoding RNA plus the 3' and 5' adaptors). At last, library quality was assessed on the Agilent Bioanalyzer 2100. The library preparations were sequenced on an Illumina Hiseq 2500/2000 platform and 50bp single-end reads were generated.