Data from: The ORFIUS complex regulates ORC2 localization at replication origins (Flow cytometry)

Hill, Sarah1

Research facility: Dana-Farber Cancer Institute

Published Jan 12, 2024 on Dryad. https://doi.org/10.5061/dryad.sn02v6x89

Data files

Jan 12, 2024 version files 107.56 MB

Flow_cytometry_data_for_Yang_and_Hill_ORFIUS_complex_manuscript.zip
107.56 MB
README.md
2.42 KB

Abstract

In this work, we use bromodeoxyuridine (BrdU)/propidium iodide cell cycle flow cytometry profiling to demonstrate that treatment of ovarian cancer cell lines with gene specific siRNAs, shRNAs, or drugs leads to minimal or no cell cycle arrest which might otherwise influence observed phenotypes. All raw FCS files for flow cytometry data presented in Figures 4E, 6G, S3A, S4C, S5B, S5F, S8C, S8D, S8E, S9C, S10C, and S12B are available here.

README: Flow cytometry data for Yang and Hill ORFIUS complex manuscript

In this work, we use bromodeoxyuridine (BrdU)/propidium iodide cell cycle flow cytometry profiling to demonstrate that treatment of ovarian cancer cell lines with gene specific siRNAs, shRNAs or drugs leads to minimal or no cell cycle arrest which might otherwise influence observed phenotypes. All raw FCS files for flow cytometry data presented in Figures 4E, 6G, S3A, S4C, S5B, S5F, S8C, S8D, S8E, S9C, S10C, and S12B are available here.

Description of the data and file structure

The dataset was collected by treating different cell lines with either siRNAs or shRNAs targeting specific genes or drugs which perturb replication origins, treating with BrdU, and staining the cells with anti-BrdU antibodies and propidium iodide. The cells were then analyzed by flow cytometry. We are including here for each experiment FCS files for each individual sample run. The data is organized by Figure with one folder each for Figures 4E, 6G, S3A, S4C, S5B, S5F, S8C, S8D, S8E, S9C, S10C, or S12B. Within each of these folders are three folders with the biological replicates for each Figure. If more than one cell line or more than one gene was targeted, there may be more than one folder within the figure folder for each treatment. We also provide an Excel file with each individual experiment to explain what is in the tubes (cell line and treatment).

For example, for the folder labeled Figure 4E OV8 siBRD1 siHBO1 flow: OV8 Cells were treated with a control siRNA versus two BRD1 specific siRNAs or a control siRNA versus two HBO1 specific siRNAs, treated with BrdU, harvested, stained with anti-BrdU and propidium iodide, and analyzed on a BD Fortessa Flow cytometer in three different experiments. We provide here the individual FCS files for the control and two different BRD1 siRNA treated cells for all three experiments in folders labeled by date of experiment in the folder OV8 siBRD1, and for the control and two different HBO1 siRNA treated cells for all three experiments in folders labeled by date of experiment in the folder OV8 siHBO1. In each folder there is also an Excel file with tube labels.

Sharing/Access information

This flow cytometry data is not uploaded anywhere else.

Code/Software

No code is needed to analyze the flow cytometry data, and it can be analyzed using basic flow cytometry software such as FlowJo.

Methods

siRNA transfection: 100,000 cells were plated in either a well of a 6-well plate or a 6 cm dish on day zero. On days one and two, cells were transfected with siRNAs by mixing 10 pmol of the appropriate siRNA with Lipofectamine RNAi/Max (LifeTech Cat. # 13778150) in Opti-MEM media (Gibco Cat. # 31985070) and adding the mixture directly to the cells. Transfected cells were utilized for various assays on day three or later.

Inducible hairpin experiments: OVCAR8 cells stably expressing control or BRD1 or HBO1 specific inducible hairpins along with either empty vector or shRNA-resistant BRD1 or HBO1 were assessed. Cells were plated on day 0, treated with media containing nothing or 10ug/mL doxycycline (Fisher Cat. #BP2653-5) on day 1, and then harvested after 96 hours for BRD1 and 72 hours for HBO1 for downstream analysis.

Cell cycle flow cytometry analysis: For siRNA treated cells, cells were harvested as described below 72 hours after the first transfection at the same time a parallel plate was harvested for DNA fiber or immunofluorescence analysis. For doxycycline treated shRNA cells, cells were harvested 72 or 96 hours post-doxycycline treatment for shHBO1 or shBRD1 respectively. For drug treated cells, in Figure S12B, respective cell lines were treated with DMSO vehicle, or 1µM Olaparib, AZD6738, or XL413 for 24 hours. One hour prior to harvest, cells were treated with 10µM BrdU in warm media (Biolegend Cat. #423401). After one hour of treatment, cells were trypsinized, washed in PBS, and then fixed in ice cold 70% ethanol. The ethanol was aspirated, cells were washed in PBST, and then the cells were incubated for 15 minutes at room temperature in 2N HCl. The cells were washed in PBST and then incubated for 30 minutes at room temperature in 0.1M Na2B4O7 pH 8.5 in 1%BSA. The cells were then incubated in FITC-BrdU antibody (BD Biosciences Cat. #556028) in 1% BSA for 30 minutes at room temperature. The cells were washed in PBST. Propidium Iodide-RNAse running buffer (BD Biosciences Cat. #550825) was then added to the cells for at least 30 minutes, and the cells were analyzed on a BD LSR Fortessa flow cytometer. The experiments were performed two to three times each.