RNA Polymerase II - single-cell feature values

Published: 24 July 2023| Version 1 | DOI: 10.17632/jprfpjf6ns.1
Maija Pietilä


Data in the paper "Cellular state landscape and herpes simplex virus type 1 infection progression are connected" by Pietilä et al., 2023: - Single-cell nuclear mean intensity of RNAP II S5P and S2P in HeLa, A549 and BJ cells (Supplementary Figure 15a-f) Cells were infected with HSV-1, and after fixation markers were detected by immunofluorescence imaging. Cells from HSV-1-infected wells were classified into uninfected and infected cells (indicated by “classification_infection”) as described in the paper. Single-cell features are provided as normalised background-subtracted mean intensity values for the nucleus. Data cleanup, background subtraction and normalisation of the intensity values were applied as described in the paper. Experimental conditions are in triplicate (indicated by “well_name”).


Steps to reproduce

3,500 HeLa, 4,000 A549 or 2,500 BJ cells were seeded per well in 96-well plates and grown at 37°C and 5% CO2 for ~48 (HeLa) or 72 (A549 and BJ) h. Cells were then infected with HSV-1 in serum-free DMEM using MOI 0.3-1. 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 60 min at 37°C to allow virus internalization. Non-internalized virus was removed by washing cells twice with acid buffer (40 mM Na citrate, 135 mM NaCl, 10 mM KCl, pH 3.0) and incubation in the acid buffer for 1 min. Cells were then washed four times with warm DMEM supplemented with 10% FBS and subsequently grown at 37°C before fixation and staining. For A549 cells, no acid wash was performed to prevent cell damage. At 12 hpi, cells were fixed with 4% paraformaldehyde. After fixation, cells were washed with PBS, and free aldehyde groups were quenched with 500 mM NH4Cl. Permeabilization was done using 0.2% (v/v) Triton X-100 and 0.1% (w/v) SDS followed by washing with PBS. Cells were then blocked in 3% (w/v) BSA, and after blocking cells were incubated with the primary antibodies, washed with PBS, and then incubated with the secondary antibodies. Antibodies were diluted in 3% (w/v) BSA. After PBS washing, nuclear DNA was stained with NucBlue Fixed Cell ReadyProbes Reagent (DAPI) and total protein was stained with Alexa Fluor 647 NHS Ester (Succinimidyl ester). Samples were imaged on a confocal microscope from Olympus (IXplore SpinSR10) with a Yokogawa spinning disk using a 40×/NA0.95 air objective, two excitation lasers (405/561 and 488/640 nm), and two ORCA-Fusion sCMOS cameras (Hamamatsu) or an automated spinning-disk confocal microscope from Molecular Devices (ImageXpress Confocal HT.ai) using a 40×/NA1.15 water objective, four excitation lasers (405, 470, 555, and 640 nm), and a CMOS camera. Cells, nuclei, and viral replication compartments were segmented using maximum intensity projections. Mitotic cells and cells at image borders were removed from the dataset. Computational image analysis was performed using TissueMAPS.


Universitat Zurich


RNA Polymerase, Confocal Microscopy, Fluorescence Microscopy, Immunocytochemistry, Herpesvirus