Rnase free pellet pestle

3.5 X volumes of QIAzol reagent were added to the gradient fractions containing monosomes, and RNA was extracted and separated by 17.5% denaturing PAGE. A synthetic RNA oligonucleotide with a length of 30 nt was used as a size marker. The gel was stained by Sybr Gold (Invitrogen, Cat: S11494) at room temperature for 10 min, and a gel slice containing RNA of approximately 30 nt was excised and ground by an RNase-free pellet pestle (Fisher Scientific, Cat: 12-141-364). RNA was extracted from the gel slice by adding 500 μL of 300 mM NaAc (pH = 5.5), 1 mM EDTA, and 0.25% (m/v) SDS and mildly shaking overnight at room temperature (Ingolia et al., 2012 (link)). The gel granules were excluded using Spin-X tube filter (Millipore, Cat: CLS8160) and the RNA was then concentrated by ethanol precipitation, dissolved in water, and stored at −80°C. 5′ phosphorylation and 3′ dephosphorylation were performed with T4 PNK (NEB, Cat: M0201S) following the manufacturer’s instructions, and the products were subjected to phenol/chloroform extraction and ethanol precipitation. cDNA libraries were constructed using QIAseq miRNA Library Kit (Qiagen, Cat:331505 & 331595) following the manufacturer’s instructions, except that the amplifying PCR was conducted with 9-12 cycles, and sequencing was performed using Illumina NextSeq 500 system.