DOI: 10.5061/dryad.sn02v6x89
title: Data from: The ORFIUS complex regulates ORC2 localization at replication origins (Flow cytometry)

description: 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: 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.  <strong>siRNA
 transfection: </strong>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.
 <strong>Inducible hairpin experiments:</strong>
 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. <strong>Cell cycle flow cytometry
 analysis:</strong> 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.
description: Any flow cytometry analysis software will open these FCS files.
 This would include FACS Diva or FlowJo.
description: # 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.