smFISH + 4i experiment – single-cell feature values and summaries
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: - Single-cell feature values: HeLa_smFISH_4i_cellular_features.csv.gz (Figure 1e, 2b, 2c, 2d, 2e, 3, 5b, 5c, 6a, 7a, 7c, 7d, 7e, 7f, 8, and Supplementary 3e, 3f, 4b, 6, 7, 9a, and 9d) and HeLa_smFISH_4i_viral_features.csv (Figure 3) - Single-cell spot count of HSV-1 UL19 and UL29 transcripts in HeLa cells: HeLa_smFISH_4i_UL19_spotCounts.csv (Figure 1c left UL19) and HeLa_smFISH_4i_UL29_spotCounts.csv (Figure 1c left UL29) - Summary of HSV-1 UL19 or UL29-expressing HeLa cells: HeLa_smFISH_4i_UL19_UL29_expressingCells.csv (Supplementary Figure 1a left) - Summary of ICP5 virion-containing HeLa cells: HeLa_smFISH_4i_virions_percentage.csv (Supplementary Figure 3b) - Summary of infected HeLa cells: HeLa_smFISH_4i_infectedCells.csv (Supplementary Figure 3c) HeLa cells were mock infected or infected with HSV-1 and fixed at different time points (indicated by "timePoint"). After fixation, the transcripts were detected by single-molecule RNA fluorescence in situ hybridization (smFISH) and viral and cellular markers by iterative indirect immunofluorescence imaging (4i). Single-cell feature extraction, and classification of cells were applied as described in the paper. Two or four replicates were used for mock and HSV-1 infection, respectively (indicated by “well_name”). Single-cell intensities are provided as corrected and background-subtracted or corrected, background-subtracted, and normalised mean and sum intensity values for the nucleus, cytoplasm, and whole-cell, as appropriate. Single-cell textures are provided as corrected and normalised texture values for the nucleus, cytoplasm, and whole-cell. Other single-cell features are provided with or without normalisation. Data cleanup, correction, background subtraction, and normalisation 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 then 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. smFISH was performed using ViewRNA high-content screening assay and signal amplification kits according to manufacturer’s instructions with some modifications. Protease treatment was not performed. Cells were incubated with the gene-specific probe sets for 3 h at 40°C, washed with the wash buffer, incubated with the PreAmp probes for 1 h at 40°C, washed with the wash buffer, incubated with the Amp probes for 1 h at 40°C, washed with the wash buffer, incubated with the Label probes for 1 h at 40°C, and washed with the wash buffer. Nuclear DNA was stained using DAPI. After the imaging, the smFISH signal was removed using the elution buffer (0.5 M L-glycine, 3 M urea, 3 M guanidinum chloride, 70 mM TCEP-HCl, pH 2.5), and 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. 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, nuclei, and cytoplasm were segmented 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.