title: GSM5231638: VT3797_rep_C; Caenorhabditis elegans; OTHER
library_construction_protocol: Concentrated lysis buffer was add to each frozen sample to final concentration of 20 mM Tris-HCl (pH = 7.4), 150 mM NaCl, 5 mM MgCl2, 0.5X Protease Inhibitor (Sigma, Cat:P2714), 1 mM DTT, 0.1 mg/ml cycloheximide (Millipore, Cat:C4859), 1% (v/v) Triton X100 and 5 U/ml Turbo DNase (Invitrogen, Cat:AM2238), and worm pellets were kept on ice until fully thawed. Suspended worms were transferred to 400 µm silica beads tube (OPS Diagnostic, Cat: PFAW-400-100-04) and lysed in bead beater homogenizer for 3 min at 4 °C. Lysates were then centrifuged at 25,000 rcf for 10 min at 4 °C, and supernatants were collected. To generate monosomes, RNase I (Invitrogen, Cat: AM2294) was added to a final concentration of 0.2 U per µl of harvested worm pellet. The digestion was incubated at room temperature for 40 min with gentle rotation and then quenched by adding SUPERase RNase inhibitor (Invitrogen, Cat: AM2694) at 4 U per RNase I unit. The lysates were then loaded onto 5-40 % (m/v) sucrose gradients prepared with lysis buffer without Triton X100 and centrifuged at 32,000 rpm for 3 hrs at 4 °C in an SW41Ti rotor (Beckman Coulter, Cat:331362). The sucrose gradients were fractionated using BR-188 Density Gradient Fractionation System with 60% (m/v) sucrose as chase solution, and monosome fractions were collected according to OD254 profiles. 3.5 X volumes of QIAzol reagent was added to the gradient fractions containing monosomes, and RNA was extracted and separated by 17.5 % denaturing PAGE. A synthetic RNA oligonucleotide with the 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. 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 oC. 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. 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 perform using Illumina NextSeq 500 system.