GSM7993211: null.rep3.riboseq; Caenorhabditis elegans; OTHER
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 NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB E7300) following the manufacturer's instructions,  and sequencing was performed using Illumina NextSeq 500 system.