smFISH of UL19, UL29, EEA1, ERBBB2, RELA and HPRT-1 – single-cell feature values
Description
Data in the paper "Cellular state landscape and herpes simplex virus type 1 infection progression are connected" by Pietilä et al., 2023: - HSV-1 transcript counts: HeLa_UL19_spotCounts_MOIs.csv (Figure 1c right UL19, Supplementary Figure 1b) and HeLa_UL29_spotCounts_MOIs.csv (Figure 1c right UL29, Supplementary Figure 1b) - Summary of HSV-1 UL19 or UL29-expressing HeLa cells: HeLa_HSV1_MOIs_infectedCells.csv (Supplementary Figure 1a right) - Cellular transcript counts: HeLa_EEA1_spotCounts.csv, HeLa_ERBB2_spotCounts.csv, HeLa_HPRT1_spotCounts.csv, and HeLa_RELA_spotCounts.csv (Supplementary Figure 1b) - Single-cell feature values: HeLa_smFISH_IF_mlr.csv (Supplementary Figure 1c) HeLa cells were mock- or HSV-1-infected (indicated by "condition"). After fixation, HSV-1 UL19 and UL29 and cellular HPRT-1, EEA1, ERBB2, and RELA transcripts were detected by single-molecule RNA fluorescence in situ hybridization (smFISH). Transcript quantification, single-cell feature extraction, and classification of cells were applied as described in the paper. Five replicates were used (indicated by “well_name”). Single-cell intensities are provided as background-subtracted, corrected, and normalised mean and sum intensity values for the whole-cell, nucleus, and cytoplasm, as appropriate. Other single-cell features are provided as normalised values. 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 6 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. Cells were also 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.