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

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.

Methods
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. 

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.