Superase inhibitor

About 1 × 10⁷ cells were used for RNA immunoprecipitation (RIP). Cell lysates were prepared with lysis buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 1 mM EDTA; 1% Triton-X100; 1 × EDTA-free cOmplete protease inhibitor (Roche); 0.2U/μl SUPERase inhibitor (Ambion)). Two sets of 5% input were kept for protein and RNA analyses. For DHX9 RIP, lysates were pre-cleared with 3 μg rabbit IgG (Cell Signaling Technology #2729) and 30 μl Protein A Dynabeads (Invitrogen) on a rotating platform for 1 h at 4°C. Pre-cleared lysates were incubated with 5 μg rabbit IgG (Cell Signaling Technology #2729) or rabbit anti-DHX9 antibody (Abcam ab26271) at 4°C for overnight, on a rotating platform. Protein A Dynabeads (50 μl) were added to each tube and incubated at 4°C for 4 h on a rotating platform. The beads were washed thrice with 1 × TBS (50 mM Tris, pH 7.5; 150 mM NaCl). Ninety percent of the beads were used for RNA analysis. The remaining 10% was for western blot.
RNA extraction was done using RNeasy Mini kit (Qiagen) and cDNA was generated using Advantage reverse transcription kit (Clontech Laboratories), as per manufacturers’ protocols. GoTaq DNA polymerase (Promega) was used for qPCR. Enrichment was expressed as % input using the formulae: 100 × (2^{(-ΔCt[Normalized RIP])}). Refer to ‘Supplementary Table S4’ for qPCR primers used.

HEK293T, HEK293T(3xFLAG-NRDE2), and HEK293T(NRDE2-GFP) cells were grown to confluency in 10 cm dishes, washed once in cold PBS, and lysed on ice for 5 min in 1× nondenaturing lysis buffer (Cell Signaling Technology #9803) with freshly added HALT protease inhibitor (Thermo Fisher #78430). Lysates were centrifuged at 14,000g for 10 min and precleared with Protein G Dynabeads (Thermo Fisher #10003D) for 1 h at 4°C. Five microliters of anti-FLAG M2 (Sigma F1804) or 1 µL of anti-GFP (Abcam ab290) was added per IP (equivalent of one 10 cm dish) and rotated overnight at 4°C. The next day, Protein G Dynabeads were added and rotated for 2 h at 4°C. Beads were washed five times in lysis buffer for 5–10 min each at 4°C. Protein was eluted in Laemmli sample buffer (Bio-Rad) with 10% 2-mercaptoethanol and subjected to analysis by either western blotting (see above) or mass spectrometry. For RNA immunoprecipitation, the same procedure was used except all incubations were performed in the presence of Superase Inhibitor (Invitrogen AM2694), and RNA was eluted from beads with TRIzol reagent and isolated with Direct-zol RNA Miniprep Plus spin columns (Zymo Research).

One hundred pmol of biotin-TEG-FERM₄₁ (~1.3 μg), biotinylated PRRX2 WT eRNA (~10.6 μg), biotinylated PRRX2 MT eRNA (~10.6 μg), and biotinylated GFP RNA (~23 μg) were pre-folded in Folding Buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 50 mM KCl, 5 mM MgCl₂) by heating the mixture at 65°C for 5 min, followed by gradual cooling to room temperature. A 950 μL reaction in Reaction Buffer [25 mM Tris-HCl pH 7.4, 150 mM KCl, 0.5% NP-40, 10 mM MgCl₂, 2 ng/μL of poly(I:C) (Sigma, P1530), 0.5 mM DTT, 1× complete protease inhibitor cocktail, 100 U of SUPERase inhibitor (Invitrogen, AM2696)] was assembled by combining pre-folded RNA with 1.5 mg of nuclear lysate and incubated for 30 min at room temperature with rotation. In the meantime, 50 μL of M-280 streptavidin Dynabeads per reaction (ThermoFisher, 11205D) was pre-washed in Reaction Buffer. Fifty μL of washed streptavidin Dynabeads were then added to the reaction and incubated for an additional 30 min at room temperature with rotation. RNA-protein-Dynabead complexes were then washed 5 times with Reaction Buffer supplemented with 150 mM of NaCl for 10 min at 4°C per wash. To elute the proteins, Dynabeads were resuspended in 2× SDS Loading Buffer and boiled for 10 min. The reactions were then used for Western blotting or submitted for proteomics.

Buffer exchange for 6xHis-BCAS2 was done by using Amicon Ultra-0.5 Centrifugal Filter device (Millipore, UFC501024) with 50% glycerol twice to eliminate excess salt. Concentration was measured once again with Bradford protein assay. Before binding, 100 nM Cy5-FERM₄₁ was folded in Folding Buffer (25 mM HEPES pH 7.6, 12.5 mM KCl, 0.5 mM MgCl₂) by heating the mixture at 65°C for 5 min, followed by gradual cooling to room temperature, protected from light. The binding reaction was then assembled with the following components: purified 6xHis-BCAS2 (0, 25 ng, 250 ng, 500 ng), 10 nM pre-folded Cy5-FERM₄₁, 2 ng/μL of poly(I:C) (Sigma, P1530), 1 mg/mL bovine serum albumin (Invitrogen, AM2616), 1 mM DTT, 1× complete protease inhibitor cocktail, 0.02 U/μL SUPERase inhibitor (Invitrogen, AM2696). For cold FERM41 reactions, the FERM₄₁ was folded as described above, and added into the reaction at 10 nM, 100 nM, and 1 μM. The reactions were then incubated for 15 min at 30°C before separation with 6% native PAGE gel supplemented with 1% glycerol and resolved with 1× TBE supplemented with 1% glycerol for 45 min at 4°C. Note that no loading dye was added to the reactions to avoid changing the salt concentration. The gel was then imaged directly with ChemiDoc MP system (Bio-Rad).