This BHATTACHARYA_2022_DATA_README.txt file was generated on 2022-03-10 by Tamanash Bhattacharya General Information 1. Title of dataset: Data from: Differential viral RNA methylation contributes to pathogen blocking in Wolbachia-colonized arthropods: Incucyte data for progeny virus derived from insect cells with or without methyltransferase inhibitor. 2. Author Information: Corresponding Investigator 1: Richard W. Hardy Indiana University Bloomington, Indiana, USA Email: rwhardy@indiana.edu Corresponding Investigator 2: Irene L.G. Newton Indiana University Bloomington, Indiana, USA Email: irnewton@indiana.edu Co-Investigator 1: Tamanash Bhattacharya Fred Hutchinson Cancer Research Center, Washington, USA Co-Investigator 2: Liewei Yan Washington University at St. Louis, Missouri, USA Co-Investigator 3: John M. Crawford Indiana University Bloomington, Indiana, USA Co-Investigator 4: Hani Zaher Washington University at St. Louis, Missouri, USA 3. Date of data collection: 2018-2022 4. Geographic location of data collection: Bloomington, Indiana, USA 5. Funding sources: This work was supported by NSF award to ILGN and RWH (MTM2025389), NIH R01 to ILGN (R01AI144430), NIH R21 to RWH (R21AI153785). 6. Recommended citation for this dataset: Bhattacharya et al. (2022), Data from: Differential viral RNA methylation contributes to pathogen blocking in Wolbachia-colonized arthropods: Incucyte data for progeny virus derived from insect cells with or without methyltransferase inhibitor, Dryad, Dataset Data Overview: 1. Description of Dataset: This dataset contains results from live-cell imaging experiments performed using Incucyte S3 (Essen Biosciences, USA). Experiment 1 (Datasheets 1 and 2) Growth of fluorescent reporter viruses in mosquito (C710) cells were monitored using Incucyte live cell analysis system (Essen Biosciences, USA). CHIKV (CHIKV18125-capsid-mKate) virus stocks were generated from C7/10 cells, either with or without Wolbachia by infecting naïve cells with the virus at an MOI of 10. Serum-free media was used for downstream virus purification. Media containing virus was collected five days post-infection. Virus stocks were subsequently purified and concentrated by ultracentrifugation (43K for 2.5 h) over a 27% (w/v) sucrose cushion dissolved in HNE buffer. Viral pellets were stored and aliquoted in HNE buffer before being used for all subsequent experiments. Cells were grown under standard conditions as described earlier under 5% ambient CO2 at 27ºC . Cells were plated to 75-80% confluency in 96-well plates to allow distinct separation between adjacent cells and preserve cell shape for optimal automated cell counting. Inhibition of Aedes DNMT2 activity in C7/10 cells was achieved using DNA cytosine methyltransferase inhibitor 5-deoxy-azacytidine (DAC-5, Sigma-Aldrich). Aedes albopictus C7/10 cells were treated overnight with media containing either 5μM inhibitor diluted in Dimethyl sulfoxide (DMSO) or DMSO alone. Cells per well were imaged and averaged across four distinct fields of view, each placed in one quarter of the well, every 2 hours over the course of the infection. For every sample, total fluorescence generated by cells expressing the red fluorescent reporter mKate was calculated and normalized by the cell number. A manual threshold was set to minimize background signal via automated background correction at the time of data collection. Following acquisition, data was analyzed real-time using the native Incucyte ® Base Analysis Software. Experiment 2 (Datasheet 3) Viability of Aedes albopictus C7/10 cells with and without Wolbachia was assessed using Cytotox Green Reagent (IncuCyte) over 48 hours following the addition of DMSO and MTase inhibitors 5-AZAC and DAC5. The data should be interpreted as follows: Experiment 1 The dataset is divided into two excel sheets, Sheet 1 representing target cells colonized by Wolbachia and Sheet 2 representing target cells free of Wolbachia. Each row represents a different time point (h) ranging from 0-48hpi. Rows represent experimental conditions (grouped in biological triplicates) used to test the replication of CHIKV-mKate reporter viruses derived from Wolbachia-free C710 cells (W- virus) or Wolbachia-colonized C710 cells (W+ virus). The experimental conditions represented here include C710 cells that were treated with control (DMSO) and Methytransferase-inhibitor (DAC5). Sheet 1: Infection in Wolbachia-colonized Cells Variables = 3 (Time, Inhibitor, Virus Source) Biological Replicates = 3 Values represent average number of virus positive cells/image Sheet 2: Infection in Wolbachia-free Cells Variables = 3 (Time, Inhibitor, Virus Source) Biological Replicates = 3 Values represent average number of virus positive cells/image Experiment 2: This experiment was meant to identify interactive effects between the AZA or DAC treatment and Wolbachia mediated pathogen blocking as well as cell viability defects resulting from the treatment. Mosquito cells (C710) with (wStri) or without Wolbachia (Yet) are included. Raw data from the Incucyte is included. It is divided over two spreadsheets - measurements from 24 hours and 48 hours are included. Sheet 3: 4 wells of C7Tet with equal volume (to volume of AZA/DAC diluted in DMSO) DMSO added 2 wells of C7Tet with 5uM AZA 2 wells of C7Tet with 5uM DAC 4 wells of C7wStri with equal volume (to volume of AZA/DAC diluted in DMSO) DMSO added 2 wells of C7wStri with 5uM AZA 2 wells of C7wStri with 5uM DAC Each well had 9 separate images taken at each time point. CyTOX was added to each well to give us the green count for dead cells. Sheet 4: 4 wells of C7Tet with equal volume (to volume of AZA/DAC diluted in DMSO) DMSO added 2 wells of C7Tet with 5uM AZA 2 wells of C7Tet with 5uM DAC 4 wells of C7wStri with equal volume (to volume of AZA/DAC diluted in DMSO) DMSO added 2 wells of C7wStri with 5uM AZA 2 wells of C7wStri with 5uM DAC Each well had 9 separate images taken at each time point. CyTOX was added to each well to give us the green count for dead cells.