We have now performed experiments in HT29 and HepG2 cells to test mitigation/synthetic lethality between…
We finished our first titration of remdesivir and HCQ, and, as is so typical, the conclusion was that we need to do it again. That’s not the full story though. We did determine optimal doses for our RNA-seq experiment, for which we have now already harvested cells, and we narrowed down the dose range for our CRISPR screen.
To recap, we had two goals for our drug titration:
1. RNA-seq: Determine a dose of remdesivir and HCQ that should NOT yield cell death within 8-24 hours (our two RNA-seq harvest timepoints) but should be high enough to affect cell physiology and gene expression.
2. CRISPR screen: Determine a dose of remdesivir and HCQ that leads to 50% cell survival within a 4-6 day time window.
For the RNA-seq dose, we used information from visual inspection of cells one and two days after treatment as well as flow cytometric live/dead analysis two days after treatment. Our chosen doses are noted in the 032520_drugtitration_experiment_0330summary and 033020_Rem_HCQ_RNAseq_experiment files. Note in the RNA-seq file that, in spite of choosing doses with no visually observed toxicity at day 1, many drug-containing conditions have lower RNA concentrations, suggesting effects on cell health. If our RNA-seq data is swamped by non-specific pro-apoptotic gene expression, we may have to repeat with lower doses.
For the CRISPR screen, the flow cytometry at day 5 (see 032520_drugtitration_0330d5FACS_results and 032520_drugtitration_experiment_0330summary) is our most reliable guide since it bears most in common with our actual proposed experiment. For some conditions, we found a dose with ~50% survival. For others, the drug was too toxic at the lowest dose we tested (remdesivir for the two liver cell lines, HCQ for HepG2) or fell in between tested doses (HCQ for HT29 cells). Thus, we are redoing these titrations in experiments whose protocols are also on the website (033020_Remdesivir_titration_v2 and 033120_HCQ_HepG2_HT29_titration_v2).
This won’t slow down our CRISPR screen timeline though. We are currently titrating the Brunello lentiviral libraries, and once we know the proper lentiviral dose and have enough cells expanded (later this week), we will begin our CRISPR screen. We will have our second round of drug titrations done by early next week, just in time to add drugs to our CRISPR library KO cell lines.
So, we have a busy week of preparing RNA-seq libraries for Nextseq (hopefully later this week), continuing the drug and lentivirus titrations, and if all goes well starting our CRISPR screens.