GSM1689763: wt_hrde-1_IP; Caenorhabditis elegans; RIP-Seq
150 L1 or 50 L2 C. elegans larvae were washed in M9 buffer (22nM Na2HPO4, 33 mM KH2PO4, 86 mM NaCl, 1 mM MgSO4) and digested in lysis buffer (200mM NaCl, 100 mM Tris pH 8.5, 50 mM EDTA, 0.5 % SDS, 200 µg/mL Prot-K) for 3h at 65°C followed by 15 min at 95° C to denaturate the Prot-K. Lysate was then incubated with DNase I (NEB) for 30 min at 37° C. Total RNA was then isolated using TRIZOL-LS (Life Technologies) using the manufacturer instructions and dissolved in 8 µL of H2O. For RIP-seq, HRDE-1 was precipitated from synchronized adult worms. 50 µL of packed worms were washed with M9 and sonicated in 200 µL of lysis buffer (25 mM Tris pH7.5, 150 mM NaCl, 1.5 mM MgCl, 1 mM DTT and cOmplete mini, EDTA free (Roche, 1 tablet per 100 mL) 5 times 30 sec ON, 30 sec OFF at the maximum energy setting. Lysates were centrifuged at 21000G for 5 min. 50 µL of the supernatant was used as input and 100 µL was incubated with 1 mg of anti-HRDE-1 antibody (Ashe et al. 2012) and 10 µL of Dynabeads Protein G (Life Technologies) for 2 hours. The beads were washed 5 times 10 min with wash buffer  (25 mM Tris pH7.5, 150 mM NaCl, 1.5 mM MgCl, 1 mM DTT and cOmplete mini, EDTA free (Roche, 1 tablet per 100 mL, 0.2 % Triton X-100). RNA was extracted from both input and IP with TRIZOL-LS (Life Technologies) using the manufacturer instructions and dissolved in 8µL of H2O. All 8uL of total RNA was treated with 5 U of tobacco acid phosphatase (Epicenter) at 37°C for 2 h to digest 5’ tri- and di-phosphates to mono-phosphates. RNA was size-selected between 15- to 35-nt on 15% TBE-urea gel. Gel-purified RNA was eluted overnight in 300 mM NaCl and then precipitated with 100% isopropanol and Glycoblue for 1 h at -20°C. The pellet was washed once with 75% ethanol and dissolved in nuclease-free water. Then, this purified fraction was confirmed by Bioanalyzer Small RNA chip (Agilent). Library preparation was based on the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England BioLabs) with slight modification. In brief, small RNA was first ligated to the 3’ adapter and then the 5’ adapter, both of which contained 4 random bases and were chemically synthesized by Bioo Scientific. Adapter-ligated RNA was reverse-transcribed and PCR-amplified for 14 cycles using index primers. The PCR-amplified cDNA construct was purified using AMPure XP beads (Beckman Coulter). The purified PCR reaction was checked on the Bioanalyzer using High Sensitivity DNA chip (Agilent). Size selection of the small RNA library was done on LabChip XT instrument (Perkin Elmer) using DNA 300 assay kit. Only the fraction containing 140-165 bp was pooled in equal molar ratio. The resulting 10 nM pool was denatured and diluted to 10 pM with 5% PhiX spike-in and sequenced as single-read on HiSeq 2500 (Illumina) in rapid mode for 50 cycles using on-board cluster generation. Sample “L1 N2” was sequenced as single-read on Miseq (Illumina). After demultiplexing, on average 35 million passing filter reads were obtained per sample.
GEO Accession
GSM1689763