DAY 1: Students will begin the project by using the IGV to browse genomic data previously generated by the Center on 12 cancer cell lines (6 lung cancer and 6 melanoma). Next, students will develop hypotheses regarding which genomic aberrations may render these cancer cells sensitive to the depletion of individual genes. For instance, students may hypothesize that lung cancer cells with mutated KRAS may be addicted to KRAS expression for proliferation and survival, melanoma cells with mutated BRAF may depend on BRAF expression for viability, or lung cancer cells with activated EGFR (via mutation or measured by autophosphorylation) may depend on EGFR for survival. Worksheets and lectures given by the facilitators will help to guide data analysis and hypothesis generation. Students will select two cell lines to perform RNAi experiments to test their hypotheses. One cell line will serve as a control, thus reinforcing critical elements of experimental design and the scientific method.
DAY 2–5: Students will then learn how to culture their selected cancer cell lines in the laboratory over the course of the next 4 days, in preparation for their RNAi experiments. They will measure the proliferation rates of their chosen cell lines and create a proliferation curve for each of their cell lines. Further, they will carry out three control experiments required for their RNAi screen. The first of these involves determining the optimal dose of puromycin required to kill the cells. This measurement is critical since they will use this dose of puromycin to select positively infected cells (shRNA vector encodes puromycin resistance) in their RNAi experiment. The second control experiment will help the students learn how sensitive their cancer cells are to lentivirus (sample of lentivirus encoding inert control shRNA will be provided). Since cancer cell lines have differential sensitivity to lentiviral infection, selecting an optimal dose of lentivirus for their RNAi screen minimizes non-specific toxicity (due to the VSVg pseudotyping of the shRNA-encoding lentiviral particles). Finally, the students will plate different numbers of cells in 96-well plates to determine the number of cells required to achieve 20% confluence, as desired for their RNAi screen.
DAY 6–7: Students will plate their cancer cells into 96 well plates using multichannel pipetmen. On the following day, students will use robotic methods to infect each plate of cells with a pre-made plate of lentivirus encoding 5 shRNAs targeting each of 15 candidate genes (KRAS, NRAS, BRAF, EGFR, PIK3CA, SRC, others) plus 20 control shRNAs targeting irrelevant transcripts.
DAY 8–11: Students will add their pre-determined amount of puromycin to cells to select for positively infected cells. Two days after selection, students will perform the Cell-Titer Glo assay to measure the relative proliferation and viability in each well. Also during these days, students will learn how to carry out data analysis of arrayed RNAi screening data.
DAY 12–14: Students will use a plate reader to measure the viability of cells in each well. They will convert these raw measurements to normalized scores and compare to control measurements obtained via infections with control shRNA constructs. They will analyze the data from their experimental and control cell lines to determine if their data supports or does not support their hypotheses. On the final day of this course, students will compile their data in a Powerpoint presentation and present to the other students in the course. At the conclusion of the course, students will complete a questionnaire aimed at continuous course improvement