title: GSM3578002: WT_CRISPR_WAGO-1-FLAG_input_sRNA-seq; Caenorhabditis elegans; OTHER
library_construction_protocol: Synchronous population of 40,000 worms were harvested at 48 hours post hatching and suspended in the extraction buffer (50 mM Tris, 300 mM NaCl, 1 mM EDTA, 0.1% NP40, protease inhibitor cocktails (Fermentas)) 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. 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 1 hour at 4°C. After four washes with RIP washing buffer (50 mM Tris, 500 mM NaCl, 1 mM EDTA, 0.1% NP40, protease inhibitor cocktails (Fermentas)), the beads were resuspended in 750 µL of TRI Reagent (MRC, Inc.) to extract the immunoprecipitated RNAs. 750 µL of TRI Reagent (MRC, Inc.) was also added to 10% of protein extract before the immunoprecipitation (Input). RNA extracted from IPs and Inputs were size seleceted 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) or Illumina MiniSeq platform using MiniSeq High Output Reagent Kit 75-cycles (FC-420-1001).