Lsm880 airyscan system

Confocal images were acquired with a Zeiss LSM 880 Airyscan system.
For measuring the distance of short genomic intervals to the NP (shown in Fig. 1d, Table 1, and Table 2), a single image was taken for a nucleus if it had at least one distinct green and red signal spots at the same focal plane. The distance of a signal spot to the NP was recorded as the distance between its estimated barycenter and the edge of DAPI staining. See Additional file 1: Figure S15 for an example.
Chromosome painting data (shown in Fig. 4) were acquired as z-stack. For each image, we used ImageJ [70 (link)] to determine nuclear boundary, while signals in green and red channels were extracted from the corresponding image files (Additional file 1: Figure S2). We found that the nuclear part close to the glass slide tended to become flattened, probably due to capillary forces. To reduce possible errors in distance calculation in this area, the bottom two images of each nucleus were not included so that our data analyses were only applied to a subset of the nuclear space. As nuclei landed on slide randomly, FISH signal distribution along the z-axis is independent from distance to the nuclear boundary. Such data exclusion, in principle, should not affect our conclusion concerning signal distribution in the remaining part of the nucleus.

Chick embryos [between embryonic day 3 (E3) to E4] were fixed in 4% formaldehyde and dehydrated in 30% sucrose overnight. These were then mounted in 1.5% Luria-Bertani (LB) agar, dehydrated in 30% sucrose again, and then snap frozen on dry ice. Cryosections of 20-μm thickness were then collected using a Leica CM3050s cryostat and then processed for immunofluorescence. For immunofluorescence, samples were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) and blocked in 1% donkey serum (Sigma-Aldrich). Primary antibodies were used at the following dilutions: Tuj1 (80120, BioLegend) 1:1000, Arl13b (17711-1-AP, Proteintech), Smo (20787-1-AP, Proteintech) 1:50, IFT88 (13967-1-AP, Proteintech) 1:200, and GPR161(13398-1-AP, Proteintech). All secondary antibodies were Alexa Fluor conjugates (Life Technologies) and used at 1:500. Images were acquired using a 60× 1.40 numerical aperture (NA) objective on a Zeiss Cell Observer Z1 microscope system (Carl Zeiss). 3D images of the primary cilium were acquired using a Zeiss LSM 880 Airyscan system equipped with a 60× 1.40 NA objective.

Confocal images were acquired with a Zeiss LSM 880 Airyscan system. For each nucleus, z-stack images (with 0.22 μm thickness for each optical sectioning) were taken with a 63× objective lens. The detection of DAPI and Alexa Fluor® 488 was according to the following settings: laser power = 1.3% for 405 nm and 0.67% for 488 nm; pinhole = 1 AU; filter = BP 420–480 + BP 495–550; master gain = 700; digital gain = 2. Same parameter settings were applied to all types of nuclei for image acquisition. Because each A. thaliana nucleus has two copies of target genomic DNA, it is not possible to determine the volume occupied by each copy if they overlap in space. Thus, for ease of image processing, we only recorded images of A. thaliana nuclei displaying two distinct clusters of FISH signals. Image processing was done with Fiji software and images were finally assembled in Photoshop. The volumes occupied by the probed genomic region in different nuclei were approximated according to the sum of areas of filtered FISH signals in z-stack images (Additional file 1: Figure S20). To identify pixels with FISH signal in each z-stack image, a threshold value of 25 in the green channel was used throughout.

To fluorescently stain polytene chromosomes, we first washed them with PBS with 0.3% TritonX-100 (PBT). We then blocked them for 10 min with normal goat serum, and incubated them in with a primary antibody solution for 1 h and 20 min at room temperature. The rabbit anti-GFZF primary antibody was developed by Covance against the target RRDVAEPAKGAQPDC. For our primary antibody concentrations, we used the following: rabbit anti-GFZF 1:200, rat anti-HMR 1:20 (Thomae et al. 2013 (link)), mouse anti-HP1 (DSHB C1A9), and chicken anti-GFP 1:200 (Abcam 13970). We washed the samples six times with PBT before incubating them in a secondary antibody solution for 1 h and 20 min at room temperature. All secondary antibodies were generated from goats and used at a concentration of 1:1,000. We washed away the secondary antibody with three washes of PBT. To stain DNA, we incubated the samples with Hoechst 32258 (Thermo Fisher) at 1:1,000 in PBT for 4 min. We washed twice more with PBS before mounting the samples under glass coverslips in Fluoromount-G. We imaged all our samples using the Zeiss LSM880 Airyscan system, and processed all images using the Zen software from Zeiss and the Fiji package in ImageJ.

Fixed SARS-CoV-2-infected HNE ALI cultures were fixed for 30 min in 4% formaldehyde in PBS, washed and stored in PBS. Filters were excised from plastic supports. After blocking with 5% normal donkey serum (017-000-121, Jackson ImmunoResearch) in immunofluorescence (IF) buffer (3% BSA and 0.1% NP-40 in PBS) for 1 hour at room temperature, cells were incubated with primary antibody in IF buffer for overnight at 4°C, followed by rinsing with IF buffer five times. The samples were then incubated with fluorescent-labeled secondary antibody and phalloidin (A30107, Invitrogen) in IF buffer for 1 hour at room temperature, followed by rinsing with IF buffer five times. After nuclear staining with 4, 6-diamidino-2-phenylindole (DAPI) in PBS, filters were mounted with Fluoromount-G (0100–01, SouthernBiotech) onto glass slides. The samples were visualized using a LSM880 Airyscan system (Carl Zeiss) with a Plan ApochromatX60 objective. Infection ratio was calculated as the (number of SPIKE or nucleocapsid protein positive cells)/(total cell number counted with DAPI). Cell count function in Image J was used for cell counting.