MCU data
Description
Data from the smFISH + 4i experiment in the paper "Cellular state landscape and herpes simplex virus type 1 infection progression are connected" by Pietilä et al., 2023: - Pixel coordinates within single cells and their multiplexed cell unit (MCU)-identity (Figure 4f): mcu_pixel_coordinates_mcuIDs.csv.gz - Summary of normalised 4i marker intensities in MCUs (Figure 4a): mcu_marker_intensities.csv - Summary of MCU occupancy (Figure 4b): mcu_occupancy.csv - SPS values of MCUs (Figure 4f): mcu_sps.csv - Summaries of UMAP coordinates of MCUs (Figure 4c,d): mcu_umap_allCells.csv and mcu_umap_infectedCells_12h.csv - Single-cell features to cluster infected cells into subpopulations (Figure 4e): mcu_leiden_infectedCells_12h.csv - Single-cell morphology features of cell subpopulations (Figure 4g): mcu_leiden_morphology_features.csv HeLa cells were infected with HSV-1, fixed at different time points (indicated by "timePoint"), and iterative indirect immunofluorescence imaging (4i) was performed as described in the paper. Cells were classified into uninfected cells without infected neighbors, uninfected cells with infected neighbors, and infected cells (indicated by “classification_infection”) as described in the paper. Single-pixel intensities are provided as corrected and normalised intensity values. Single-cell morphology features are provided as normalised values. MCU construction from single-pixel intensities, correction, normalisation, and Leiden clustering were performed as described in the paper.
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Steps to reproduce
3,500 HeLa cells were seeded per well in 96-well plates and grown at 37°C and 5% CO2 for ~48 h. Cells were then infected with HSV-1 in serum-free DMEM using MOI 0.3. Cells were incubated with the virus for 30 min at 4°C, and then unbound virus was removed by washing cells with warm DMEM supplemented with 10% (v/v) FBS. Cells were then incubated for 60 min at 37°C, and non-internalized virus was removed by washing cells with acid buffer (40 mM Na citrate, 135 mM NaCl, 10 mM KCl, pH 3.0). Cells were then washed with warm DMEM supplemented with 10% (v/v) FBS and grown at 37°C. At 1.5-12 hpi, cells were fixed with 4% paraformaldehyde. After fixation, cells were washed with PBS, and free aldehyde groups were quenched with 0.1% (w/v) NaBH4. Cells were then washed with PBS and further quenched with 100 mM glycine and washed with PBS. Next, cells were permeabilized with 0.2% (v/v) Triton X-100 followed by washing with PBS. 4i was performed as previously described (Gut et al., 2018) with some modifications. Per 4i cycle, (1) cells were washed with PBS and blocked in Intercept blocking buffer supplemented with 100 mM NH4Cl, 150 mM Maleimide, and 5% (v/v) donkey serum. (2) Cells were washed with PBS and incubated with the primary antibodies. Cells were then washed with PBS and incubated with the secondary antibodies. Antibodies were diluted in Intercept blocking buffer supplemented with 100 mM NH4Cl. Cells were subsequently washed with PBS, and nuclear DNA was stained with DAPI. Then, cells were washed with PBS and, (3) imaged in the imaging buffer (700 mM N-Acetyl-Cysteine, 200 mM HEPES, pH 7.4). (4) Antibodies were eluted using the elution buffer (0.5 M L-glycine, 3 M urea, 3 M guanidinum chloride, 70 mM TCEP-HCl, pH 2.5). In the last cycle cells were stained with a total protein stain, Alexa Fluor 647 NHS Ester (Succinimidyl ester). Samples were imaged on an automated spinning-disk confocal microscope (Yokogawa CellVoyager 7000) using a 40×/NA0.95 air objective, four excitation lasers (405, 488, 568, and 647 nm), and two Neo sCMOS cameras (Andor). Cells were segmented and single-pixel intensities and single-cell features were quantified using maximum intensity projections. Mitotic cells and cells at image borders were removed from the dataset. Computational image analysis was performed using TissueMAPS.