title: GSM4761331: HRDE-1_GFP_lateL4_IP_rep1_sRNA-seq; Caenorhabditis elegans; RIP-Seq
library_construction_protocol: Synchronous populations of L1 worms were grown at 20°C on NGM plates seeded with OP50 E. coli concentrated food at a density of maximum 40,000 animals per 15 cm Petri dish. 80.000 worms were collected at the indicated developmental stage: early L4 ( 38 hours post hatching (hph)), late L4 (44hph) or Young Adult ( 48hph). Worm pellets were washed three times with M9 buffer and suspended in the extraction buffer (50 mM Tris, 500 mM NaCl, 1 mM EDTA, 1% NP40, protease inhibitor cocktails (Thermo Scientific, 1860932)) followed by 30 strokes using a metal dounce on ice. Crude protein extracts were spun at 12,000 rpm at 4°C for 10 minutes. For CSR-1 IPs, Protein concentration was quantified by the Bradford assay, and 1 mg of protein extract was incubated with 15 μl of packed Anti-FLAG M2 Magnetic Agarose Beads (Sigma M8823). For HRDE-1 IPs, 2 mg of protein extract was incubated with 25uL GFP-trap Agarose beads (ChromoTek) for 1 hour at 4°C. After four washes with IP washing buffer (50 mM Tris, 500 mM NaCl, 1 mM EDTA, 1% NP40, protease inhibitor cocktails (Thermo Scientific, 1860932)), the beads were resuspended in 750 µL of TRIzol LS reagent (Ambion) to extract the immunoprecipitated RNAs. 750 µL of TRIzol LS reagent (Ambion) was also added to 10% of protein extract before the immunoprecipitation (Input). RNA extracted from IPs and Inputs were size selected from 15% UREA PAGE gel. The library preparation was performed as essentially described by Jayaprakash et al., 2011, except that a 5' polyphosphatase (lucigen RP8092H) treatment was performed to be able to clone tri-phosphate small RNAs, and that the PAGE gel extraction after each ligation was substitute with purification by 1.8 volumes of Agencourt RNAClean XP Beads (Beckman Coulter, NC0068576) and 3 volumes of isopropanol. The multiplexed amplified libraries were further purified using PippinPrep DNA size selection with 3% gel cassettes and the following parameters for the selection: BP start (115) and the BP end (165). The purified libraries were quantified using Qubit Fluorometer High Sensitivity dsDNA assay kit (ThermoFisher, Q32851) and sequenced either on NextSeq-500 Illumina platform using the NextSeq 500/550 High Output v2 kit 75 cycles (FC-404-2005).