Optiprep medium

The virus stock was concentrated 25-fold using centrifugal protein concentrators (Amicon Ultra, 10 kDa cut-off; Merck, Poland) and subsequently overlaid on 15% iodixanol solution in 1 × PBS (OptiPrep medium; Sigma-Aldrich, Poland). Following virus concentration using iodixanol cushion (centrifugation at 175 000 × g for 3 h at 4°C), it was overlaid on 10-20% iodixanol gradient (in 1 × PBS) and centrifuged at 175 000 × g for 18 h at 4°C. Fractions (1 ml) collected from the gradient were analyzed by Western blotting, followed by immunodetection of the HCoV-NL63 nucleocapsid protein. The virus-containing fractions were aliquoted and stored at −80°C. The control cell lysate (mock) was concentrated and prepared in the same manner as the virus stock.

First, 164K pellets collected by differential centrifugation from 8–15 × 10⁷ cells were resuspended in 1.1 mL of STE buffer (0.25 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4) and transferred to the tube of SW60Ti rotor (Beckman) and mixed 1:1 with 60% (wt/vol) stock solution of iodixanol (Optiprep medium, Sigma). STE buffer diluted iodixanol solutions, 1 mL of 20% iodixanol, and 0.8 mL of 10% iodixanol were layered sequentially on the top and the tube centrifuged at 350,000× g and 4 °C for 1 h (brake off). Eight fractions of 480 μL were collected from the top of the tube. For Western blot and CCA in the downstream analyses, 320 μL and 160 μL (respectively) from each 480 μL fraction was diluted with 2 mL and 240 μL PBS (respectively) and centrifuged at 164,000× g for 30 min in TLA110 and TLA 120.1 rotors (Beckman; respectively). For Western blot and immuno-isolation in the downstream analyses, each fraction was split into two 240 μL portions, diluted with 2 mL PBS for Western blot and 560 μL PBS for immuno-isolation, followed by centrifugation in TLA110 and TLA 120.2 rotors (Beckman), respectively. The pellets were resuspended in PBS for immuno-isolation, radioimmunoprecipitation assay (RIPA) lysis buffer (Cell Signaling) for Western blot, or Quick C-Circle Preparation (QCP) lysis buffer [16 (link)] for CCA, and stored at −20 °C.

Virus-containing medium was concentrated using Amicon Ultra, 10 kDa cut-off (Merck, Warsaw, Poland) and subsequently overlaid on 15% iodixanol solution in PBS (OptiPrep medium; Sigma-Aldrich, Poznan, Poland) and centrifuged at 45,000× g for 3 h at 4 °C. Following centrifugation virus-containing fraction was overlaid on 10–20% iodixanol gradient and centrifuged at 45,000× g for 18 h at 4 °C. Fractions collected from gradient were analyzed by Western blotting to detect the CRCoV nucleocapsid protein. The virus-enriched fractions were aliquoted and stored at −80 °C.

Stock OptiPrep medium (60%) (Sigma-Aldrich) was diluted to 50, 40, 30, 20, or 10% using distilled H₂O and 900 μl of each diluted medium was layered gently into a 5 ml ultracentrifuge tube in a descending concentration from 50 to 10%. Cell lysate (500 μl each) was layered onto the top of the gradient and the tubes were centrifuged at 145,000 x g for 12 h in a SW 55 Ti rotor at 4°C. After centrifugation, fractions (500 μl each) were withdrawn from the top and 25 μl of each fraction was analyzed by Western blotting.

Mice were sacrificed by cervical dislocation. After hippocampal dissection, cell dissociation and lysis was performed by mechanical homogenization in a 2 mL dounce homogenizer (Sigma-Aldrich) filled with 1 mL Nuclei Extraction Buffer (250 mM Sucrose, 25 mM KCl, 5 mM MgCl₂, 20 mM Hepes-KOH, 65 mM β-glycerophosphate, 0,5% IGEPAL CA-630, 0,2 mM Spermine, 0,5 mM Spermidine, Proteinase inhibitors (complete EDTA-free, Roche)). Using 35 μm mesh-capped tubes, nuclei were filtered to get rid of debris. Incubation with 0.01 mM DAPI. For the clean isolation of nuclei, samples were diluted in a 22% Optiprep medium (D1556, Sigma-Aldrich) solution, and a density gradient was prepared in 15 mL centrifuge tubes as follows: 2 mL 44% Optiprep, 1 mL 22% Optiprep, 22% sample containing the nuclei, and additional 22% Optiprep to fill the tube. The gradient was centrifuged for 20 minutes at 10000 x g and the phase containing the nuclei was collected. Nuclei were then sorted in a flow-cytometer FACS Aria III (BD Bioscience) and GFP positive nuclei were selected according to the parameters defined in ref. ^{26 (link)} (see step by step analysis and channel filtering details in Supplementary Fig. 4H).