title: GSM8911495: SS142 20°C IP 3; Caenorhabditis elegans; ncRNA-Seq
library_construction_protocol: The worm pellet was resuspended at a 1:1.5 (v/v) ratio in EDTA buffer, which contained 10% glycerol, 10 mM EDTA, 30 mM HEPES, 100 mM potassium acetate, 2 mM DTT, and 0.1% NP-40, supplemented with protease, RNase, and phosphatase inhibitors: 2x cOmplete EDTA-free protease inhibitor cocktail (Roche), 1:100 SuperaseIN (Thermo Fisher), and 1:100 phosphatase inhibitor cocktails 2 and 3 (Sigma). The resuspended pellet was flash-frozen by adding it drop by drop into liquid nitrogen, and the frozen samples were stored at –80°C until lysate preparation.  The frozen samples were ground in a mortar with liquid nitrogen. Protein lysates were prepared by thawing the frozen powder, sonicating it at a low setting (3 minutes, 15 seconds ON/15 seconds OFF), and centrifuging it at 17,000 x g for 15 minutes, with all steps performed at 4°C. Once thawed, the samples were handled continuously at 4°C or kept on ice. To determine the total protein concentration, either a Lowry assay or a Bradford assay (BioRad) was used. For total sRNA sequencing, an aliquot containing 500 µg of protein was mixed with TRI Reagent (Sigma) and flash-frozen (further description in the RNA isolation step).  To sequence the sRNAs bound to WAGO-4, we performed an immunoprecipitation (IP) protocol. All the assayed strains expressed WAGO-4 tagged with 3xFLAG::GFP tag. For the IPs, 3-6 mg of total protein was used per reaction. To pre-clear the lysate, it was incubated with 20 µL of Binding Control magnetic beads (ChromoTek) at 4°C for an hour with rotation. The lysates were then incubated with GFP-Trap magnetic beads (ChromoTek) at 4°C for an hour with rotation to bind WAGO-4. Both the Binding Control and GFP-Trap magnetic beads were equilibrated with the IP buffer prior to adding the lysate. After binding to the GFP-Trap beads, the beads were washed once with a buffer containing 30 mM HEPES, 400 mM KCl, and 0.4% NP-40 and four washes with IP buffer. All washes were performed by rotating for 10 minutes at 4°C. For each replicate, 2-3 IP reactions were pooled in 200 μL of IP buffer. Next, 720 μL of TRI Reagent (Sigma) was added, and the samples were flash-frozen and stored at –80°C until RNA extraction.  RNA was isolated from the samples containing TRI Reagent (mixed with lysate for the total pool of sRNAs or with the GFP-Trap magnetic beads for the WAGO-4 bound sRNAs, as described above). Samples were vortexed at room temperature for 15 minutes. Then, 10% 1-bromo-3-chloropropane was added and the mixture was centrifuged at 12,000 × g for 15 minutes at 4°C. The aqueous phase was separated and mixed with phenol:chloroform:isoamyl alcohol (pH 4.5) in a 1:1 ratio, followed by centrifugation (12,000 × g for 15 minutes at 4°C). The clear aqueous phase was separated and mixed with 20 µg of glycogen, 0.3 M NaOAc (pH 5.2), and an equal volume of isopropyl alcohol. The RNA was precipitated at -20°C for at least 30 minutes. Samples were centrifuged at 16,000 × g for 30 minutes at 4°C, and the pellet was washed twice with 900 µL of 70% ice-cold ethanol (the centrifugation step between the two washes was at 16,000 × g for 10 minutes at 4°C). The pellet was air-dried at room temperature for 10 minutes and resuspended in 11 µL of nuclease-free water. The RNA concentration was measure using Nanodrop and up to 4 µg was used for the 5′ polyphosphatase reaction (Manufacturer). The reaction was incubated for 30 min at 37 °C and RNA was extracted using phenol:chloroform:isoamyl alcohol as described above. The air-dried pellet was resuspended with 6-7 µL nuclease free water and was used for the library preparation. Small RNA libraries were created using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs) according to the manufacturer's instructions. Starting material included 1 μg of total RNA or the entire yield of immunoprecipitated RNA. The resulting DNA libraries were size selected by excising the bands with the size range of ~135–150 bp on 8% PAGE gel (correspond to 16–30 bp RNAs). The DNA was eluted by rotating overnight in 500 μl of DNA Gel Elution buffer at room temperature. Subsequently, the DNA library was precipitated with 20 μg of glycogen 0.3 M sodium acetate (pH 5.2), and an equal volume of isopropyl alcohol; pellets were washed and dried as previously described. They were resuspended in 12 μl of nuclease free water and quantified using a Qubit HS DNA kit (Thermo Fisher). sRNA libraries were pooled in equal amounts and sequenced on Illumina Miniseq and Novaseq 6000.