Benzonase nuclease

Immunoprecipitations were performed on three biological replicates of both ORC2-GFP and ORR embryos. For each replicate, 0.5 g of embryos aged 18–24 h were collected, dechorionated, and flash frozen. Frozen embryos were ground thoroughly with a mortar and pestle in liquid N2. Ground embryos were then resuspended in NP40 Lysis Buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1% NP40, 1 mM EDTA, 1 mM EGTA, with 2X cOmplete^™ Protease Inhibitor Cocktail EDTA-free (Millipore Sigma)). The embryonic extract was treated with benzonase nuclease (Millipore #7066) at a final concentration of 30 U/mL for 30 min at 4°C. After benzonase treatment, the extract was centrifuged at 4,000 rcf for 5 min. The supernatant was then used for the immunoprecipitation.
Prior to the immunoprecipitation, GFP Trap magnetic agarose beads (Chromotek #gtma-10) were washed and equilibrated with NP40 lysis buffer. Beads were added to extract and incubated for 2 h at 4°C. After the 2 h, beads were isolated and washed with 4 times with NP40 lysis buffer. Beads were then resuspended in 2× Laemmli sample buffer (Biorad #1610737) and boiled at 95°C for 20 min to elute protein.

An AAV vector was generated using an AAV-2 Helper-Free System in accordance with the manufacturer’s protocol (Cell Biolabs, Inc. CA). A plasmid with AAV capsid serotype 9 (pAAV-RC9) was obtained from Penn Vector Core at the University of Pennsylvania. A plasmid with AAV conditional shRNA (pAAV-dsRed-Sico-shRNA), in which the shRNA was expressed only when regulated by Cre recombinase was kindly provided by Dr. Marina Picciotto, Yale University⁴⁰ . The purification method was performed with slight modifications^{62 (link)}. Briefly, AAV-293 cells (Agilent Technologies, CA) were transfected with pAAV-GFP or pAAV-conditional shRNA, pHelper, and pAAV-RC9 vector plasmids using a standard PEI (Polysciences, Inc. PA)-mediated transfection method. 72 h after transfection, cells were collected and suspended in artificial CSF solution containing 124 mM NaCl, 3 mM KCl, 26 mM NaHCO₃, 2 mM CaCl₂, 1 mM MgSO₄, 1.25 mM KH₂PO₄ and 10 mM d-glucose. Following four freeze–thaw cycles, the cell lysates were treated with Benzonase nuclease (Millipore) at 45 °C for 15 min, then centrifuged twice at 16,000×g for 10 min at 4 °C. The supernatant was used as the virus-containing solution. Quantitative real-time PCR was performed to measure the titer of the purified virus. Virus aliquots were then stored at -80 °C until used for the experiment.

Immunoprecipitation was performed as described previously^{33 (link)}. HEK293 cells stably expressing Flag-BEX2 (1 ml) and the control cells expressing the induced pcDNA5/FRT/TO vector only (1 ml) were suspended in extraction buffer (50 mM HEPES pH 7.4, 0.3 M NaCl, 0.2% NP40) and sonicated for 5 min. The cell lysates were clarified by centrifugation at 10,000g for 30 min at 4 °C. The supernatants were filtrated through a Minisart syringe filter (Sartorius) and incubated with anti-flag antibody M2 beads (40 ml, sigma-Aldrich) for 4 h in the presence of Benzonase nuclease (10 mg/ml, Millipore) at 4 °C. After washing three times with washing buffer (0.15 M NaCl, 0.1% NP-40, 50 mM HEPES pH 7.4) and once with PBS, the binding proteins were eluted in 40 ml 0.1 M glycine buffer, pH 3.0. The eluates were neutralized with 4 ml 1 M Tris–HCl buffer pH 9.5 and suspended in SDS-PAGE (poly-acrylamide gel electrophoresis) sample buffer. The samples were boiled for 5 min and resolved by SDS-PAGE. The gel was stained using a mass silver stain kit (Wako, Osaka, Japan).

Rabies viruses used for retrograde tracing (B19G-SADΔG-EGFP, B19G-SADΔG-tdTomato) were generated in-house (Osakada and Callaway, 2013 (link); Wickersham et al., 2010 (link)). Virions were amplified from existing stocks in several rounds of low-MOI passaging through BHK-B19G cells by transferring filtered supernatant, with 3 to 4 days between passages a maximum of 3 passages. Cells were grown at 35°C and 5% CO₂ in DMEM with GlutaMAX (Thermo Scientific, #10569010) supplemented with 5% heat-inactivated FBS (Thermo Scientific #10082147) and antibiotic-antimycotic (Thermo Scientific #15240–062). Media containing virions were collected at the end of the last passaging round and incubated with benzonase nuclease (1:1000, Millipore #70664) at 37°C for 30 min, followed by filtration through a 0.22 µm PES filter. Filtered supernatant was transferred to ultracentrifuge tubes (Beckman Coulter #344058) with 2 ml of a 20% sucrose in dPBS cushion and ultracentrifugated at 20,000 RPM (Beckman Coulter SW 32 Ti rotor) at 4°C for 2 hr. The supernatant was discarded and the pellet was resuspended in dPBS for 6 hr on an orbital shaker at 4°C before aliquots were prepared and frozen for long-term storage at −80°C. Unpseudotyped rabies virus titers were estimated using a serial dilution method counting infected HEK 293 T cells and quantified as infectious units per ml (IU/ml).