GSM5257767: siRNA mop1-1/mop1-1 rep1; Zea mays; RIP-Seq
Protocols were adapted from Havecker et al. (2010) using two biological replicates per genotype. Tissues were ground with liquid nitrogen and a Dounce homogenizer. Resulting powder was placed in a Falcon tube with 3 volumes of Extraction buffer (20 mM Tris HCL pH 7.5, 5 mM MgCl2, 300 mM NaCl, 0.1% NP-40, 5 mM DTT, 1% protease inhibitor (Roch Tablet), 100 units/mL RNase OUT (invitrogen)). Samples were vortexed, kept on ice 30 minutes with recurrent shaking, and centrifuged 30 minutes at 4°C, 4000 rpm. Supernatants were filtered through a 0.45µm Miracloth into a new Falcon tube, and 1 mL and aliquoted and stored at -20°C as a pre-experiment input sample. In the remaining samples, 1.5 mL aliquots were generated, and we added 5 µg of antibodies per gram of tissue. They were incubated overnight at 4°C on a rotation wheel. Magnetic beads (Dynabeads, Life technologies) were washed 3 times in Wash buffer (20 mM Tris HCL pH 7.5, 5 mM MgCl2, 300 mM NaCl, 0.1% NP-40, 1% protease inhibitor (Roch Tablet), 100 units/mL RNase OUT (invitrogen)). 80 µL of washed beads were added to each sample, and incubated on rotation wheel for 3 hours at 4°C. Beads from the samples were washed 3 times in Wash buffer, and resuspended in 500 µL. 100 µL was aliquoted and stored at -20°C for the western blot control. Wash buffer was discarded and replaced by 250 µL of Elution buffer (100 mM NaHCO3, 1% SDS, 100 units/mL RNase OUT (Invitrogen)), and tubes incubated overnight at 65°C with agitation. Supernatant was transferred to fresh tubes, 250 µL of Elution buffer was added, and the tubes incubated 15 minutes at 65°C with agitation. Samples were treated with 0.08 µg/µL proteinase K for 15 minutes at 45°C. RNAs were extracted following the recommendations from Applied Biosystems for TRI Reagent® Solution, starting by adding 1.2 mL of TRI Reagent to the samples The RIP antibodies we used were all home-made by EUROGENTEC.  AGO104: SERICKEQTFPLRQR.  AGO105: VGQFIKFDEMSETS. NEBNext Small RNA Library Prep Set for Illumina