Convergence of distinct RNA-silencing pathways on GW182/Tnrc6 [RIP-seq II]

RIP-sequencing in mouse embryonic stem cells. Experiment 3 (Samples 2-24) & 4 (Samples 25-48): Base cell line (untagged control cells): GFP_5xT71BS (mES [129S6/SvEvTac], yGlob [GFP_5xT71BS], stable transfection FLAG-Cas9::T2A::Blasticidin). HA-Tnrc6a: endogenously HA-AVI-tagged Tnrc6a. HA-Tnrc6a_Trim71KO: HA-AVI-tagged Tnrc6a, Trim71-/-. Experiment 2 (Samples 19-42): Ctrl: base cell line GFP_5xT71BS. Tnrc6a: endogenously HA-AVI-tagged Tnrc6a. Tnrc6a_Trim71KO: HA-AVI-tagged Tnrc6a, Trim71-/-. Tnrc6a_Ago2KO: HA-AVI-tagged Tnrc6a, Ago2-/-. Cells were grown in triplicates to confluency in 10 cm dishes. Cells were washed 1x with PBS and lysed with lysis buffer (25 mM Tris-HCL at pH 7.5, 100 mM KCl, 0.5% [v/v] NP-40, 1 mM EDTA, EDTA-free cOmplete miniprotease inhibitor [Roche], PhosSTOP [Roche]. RNAse inhibitors [SUPERase•In™ RNase Inhibitor, Invitrogen, AM2694] was added to all buffers. Lysates were cleared by centrifugation. Equal amounts of protein were incubated with 20 µL of Pierce™ HA magnetic beads (Thermo Scientific, 88836) for 2 h at 4°C. Beads were washed three times with 200 µL of washing buffer (50 mM Tris-HCl at pH 7.5, 150 mM KCl, 5 mM MgCl2, 0.05% NP-40, EDTA-free cOmplete miniprotease inhibitor [Roche, 11836153001], PhosSTOP [Roche]). RNA was isolated using the Norgen single-cell RNA purification kit (Norgen, 51800) with on-column DNase digestion (Norgen, 25710).