Accuri c6 sampler

Fluorescence-activated cell sorting (Sony) was performed on FNR-RFP parasites grown for a full lytic cycle with either no drug, 40 μM actinonin, 100 nM clindamycin, or 25 μM ciprofloxacin. Untagged parasites were used for gating on FNR-RFP⁻ cells. One million cells were sorted from each population and frozen at −80°C for subsequent analysis.
Flow cytometry (BD Accuri C6 sampler) was performed to count parasites and quantify FNR-RFP fluorescence. Untagged parasites were used for gating on FNR-RFP⁻ cells. At each time point, syringe-lysed cells were washed and resuspended in phosphate-buffered saline (PBS), and 10-μl fixed volumes were quantified. Samples were always resuspended in PBS directly prior to measurement in the flow cytometer.

Since miR-146a is an inflammatory associated miRNA, we decided to measure the levels of certain cytokines. The BD Cytometric Bead Array Human Th1/Th2/Th17 Cytokine kit was used to measure Interleukin (IL)-2, IL-4, IL-6, IL-10, IL17a and tumour necrosis factor-alpha (TNF-α) protein levels in a serum samples. Briefly, lyophilized standards were prepared by reconstitution and serial dilution (1:2–1:256) in assay diluent immediately before staining with Capture Beads and Phycoerythrin Detection Reagent. All serum samples were also diluted in assay diluent (1:4) before staining with Capture Beads and Phycoerythrin Detection Reagent. For the staining procedure, 50 μL of each standard and unknown sample was added to appropriately labelled sample tubes followed by 50 μL of the Human Th1/Th2/Th17 Phycoerythrin Detection Reagent and incubated (3 h, RT, protected from light). Following incubation 1 mL of Wash Buffer was added to each assay tube and centrifuged at 200g for 5 min. The supernatant from each assay tube was then carefully aspirated and 300 μL of Wash Buffer was added to each assay tube to resuspend the bead pellet. Flow cytometric data was acquired using the BD AccuriC6 Sampler counting 2100 gated events. This ensures that the sample file contains approximately 300 events per Capture Bead. Data analysis was performed using the FCAP Array analysis software.

The cells were trypsinised, washed with PBS and fixed with 70% ice-cold ethanol. For cell cycle analysis, the cells were washed with PBS and re-suspended in PI/RNase staining buffer. FACS profile were then determined and analysed using BD accuri C6 sampler.

Doxycycline inducible MCL1, BCL2L1, MARCH5, and WSB2 anchor cell lines were generated by delivering the pRDA_103 vector via a no-spin transduction. Meljuso cells were seeded in a T75 flask in a total volume of 8.6 mL of virus-containing media with polybrene at 0.5 μg mL⁻¹. Flasks were then transferred to an incubator overnight, and the virus-containing media was replaced with fresh media 16–18 h after seeding. Blasticidin selection was added 2 days post-transduction and was maintained for 14 days. Cells were then transduced with pXPR_124 (SpyoCas9-P2A-EGFP) at an MOI of ~0.5, generating a mixed population of EGFP+ and EGFP− cells. After 5 days, cells were treated with 1 μg mL⁻¹ of doxycycline to induce expression of Spyo-guide. On day 7 post infection, cells were treated with 250 nM of either S63845, venetoclax (ABT-263), A-1331852, or navitoclax (ABT-199). The fraction of EGFP-positive cells was monitored for 2 weeks by flow cytometry (BD Accuri C6 Sampler) upon every cell passage. An example gating strategy is provided in Supplementary Fig. 17.

A375 cells were transduced with pLX_311-Cas9 to generate lines stably expressing Cas9. After 2 weeks of selection with blasticidin, vectors delivering EGFP and a guide targeting PARP1 were introduced, resulting in a heterogeneous population of EGFP-positive and negative cells. Three days post-transduction with guide construct, cells were treated with varying doses of olaparib, talazoparib, niraparib, or veliparib. The fraction of EGFP-positive cells was monitored for 17 days by flow cytometry (BD Accuri C6 Sampler) upon every cell passage.

FACS analysis was carried out as in Schalbetter et al. (2015) (link): 500 μl of culture samples were re-suspended in 70% ethanol for fixing. They were then spun down and re-suspended in 1 mL of 50 mM Tris-HCl pH8 with 5 mg/ml RNaseA (Sigma-Aldrich) at 37°C overnight. Samples were pelleted and re-suspended in 5 mg/ml pepsin (Sigma-Aldrich) and 5 μl/ml concentrated HCl. They were then incubated at 37°C for 30 minutes. Samples were pelleted and washed in 50 mM Tris-HCl pH8 before being re-suspended in 50 mM Tris-HCl pH8 with 0.5 mg/ml Propidium iodide (Sigma-Aldrich) and sonicated ready for analyzing using the BD Accuri C6 sampler and analyzed using FCS express 4 flow software. FACS analysis for all the ChIP-SEQ experiments are shown in Figure S7.