GSM6364495: Whole worm, 3xFLAG-WAGO-10, input, rep2; Caenorhabditis elegans; RIP-Seq
Synchronous populations of ~100,000 L1 worms were plated on 15 cm NGM plates with ~2ml of 5x concentrated OP50 E. coli as a food source. Five of these plates were used as starting material for protein isolation. Worms were grown for 48h for L4 staged worms or 58h for young adults (worms that had transitioned to producing oocytes but not yet with embryos). Worms were washed three times with M9 buffer (22mM KH2PO4, 42mM Na2HPO4, 86mM NaCl) and one time with EDTA buffer (10% glycerol, 10mM EDTA, 30mM HEPES, 100mM Potassium Acetate). The pellet was flash frozen in a dry ice/ethanol bath. The frozen pellets were stored at -80°C. The frozen pellet was resuspended 1.5:1 (v/v) in ice-cold IP buffer (10% glycerol, 10mM EDTA, 30mM HEPES, 100mM Potassium Acetate, 2mM DTT, 0.1% NP-40) supplemented with protease and phosphatase inhibitors (1 tablet per 5ml buffer of cOmplete™, mini, EDTA-free protease inhibitor cocktail [Roche], 1:100 phosphatase inhibitor cocktail 2 [Sigma], 1:100 phosphatase inhibitor cocktail 3 [Sigma]). If the pellet was to be used for RNA purification, 1% (v/v) SuperaseIN RNAse inhibitor (ThermoFisher) was added. The pellet was homogenized using a stainless steel dounce homogenizer (Wheaton Incorporated) until intact worms were no longer visible. Extracts were centrifuged at 13,000g for 10 min at 4°C, and the supernatant transferred to a fresh tube. A Lowry assay (Lowry et al., 1951) was performed to determine total protein concentration using a Nanodrop 1800C spectrophotometer (ThermoFisher). All IPs were conducted on 5mg of total protein per reaction. Input control samples were made by taking 10% of the lysate before the addition of antibodies. For the IP of GFP tagged Argonautes, GFP-Trap_MA beads (ChromoTek) were equilibrated by washing them three times in 1ml of IP buffer. 20μl of beads were added to 5mg total protein in a reaction volume of 500μl and rotated at 4°C for an hour. The beads were then washed four times with 1ml of IP buffer, separated from the supernatant on a magnetic stand, and rotated for 10 min at 4°C between each wash. For the IP of 3xFLAG tagged Argonautes, Monoclonal Anti-FLAG M2 (Sigma-Aldrich) were bound to Dynabeads™ Protein G (ThermoFisher) or GB-Magic Protein A/G Immunoprecipitation Magnetic Beads according to manufacturer's instructions. 5μg of Anti-FLAG M2 in 200μl of PBS-T were added to 50μl of Dynabeads and rotated at room temperature for 10 min. Then IPs were conducted as described above except the whole 50μl of ANTI-FLAG M2 bound beads were used in the IP. For small RNA sequencing, three to six IP reactions were combined in 200μl of IP buffer. 800μl of Tri Reagent (Molecular Research Centre) was added and the samples were frozen at -80°C. RNA was isolated using Tri Reagent (Molecular Research Centre). Samples were mixed with Tri Reagent in a 1:4 ratio and frozen at -80°C. Samples were vortexed at room temperature for 15min and frozen again at -80°C for 15min. This was repeated for a total of three times. 100μl of chloroform was then added to the samples and centrifuged at 12,000g for 15min at 4°C. The top aqueous phase was transferred to a fresh tube. Phenol:chloroform:isoamyl alcohol (Sigma-Aldrich) was added in a 1:1 ratio and centrifuged at 12,000g for 15min at 4°C. The top aqueous phase was transferred to a fresh tube. 20μg of glycogen (Ambion) and a 1:1 ratio of isopropanol was added to the samples and incubated at -20°C for 30min. Samples were then centrifuged at 16,000g for 30min at 4°C and the supernatant was removed. The pellet was washed with 900μl of 70% ice-cold ethanol for 10min and centrifuged at 16,000g for 10min at 4°C. This was repeated twice. The pellet was then left to air dry for 10min at room temperature and then resuspended in 6-25μl of RNase free water preheated to 70°C. Small RNA libraries were prepared with the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs) following the protocol provided by the manufacturer. 1μg of total RNA or immunoprecipitated RNA was used as starting material. The resulting DNA library was visualized using 8% PAGE and bands corresponding to small RNAs of size range 16-30bp (∼135-150bp on gel) were excised. The DNA was eluted from the excised bands by rotating overnight in 500μl of DNA Gel Elution buffer at room temperature. The DNA was precipitated with 20μg glycogen (Ambion), 50μl of 3M Sodium Acetate pH 5.2 and 1 volume of isopropanol (Sigma-Aldrich) as described above and ultimately resuspended in 12μl of Ultra-Pure water. The library DNA was quantified using a Qubit HS DNA kit (ThermoFisher). Between 12-19 Libraries were pooled in equal amounts and sequenced on a HiSeq 2500 Sequencing System (Illumina).