Illumina HiSeq 2000 sequencing; Kinome-wide shRNA screen for synthetic lethality in patient-derived FANCA-deficient fibroblasts.
Kinome-wide shRNA screen for synthetic lethality in patient-derived FANCA-deficient fibroblasts.
Patient derived fibroblasts from a donor that had Fanconi anemia due to loss of FANCA. FANCA was restored to the cells via viral transduction with a plasmid encoding wild-type FANCA.  Isogenic control line was generated by viral transduction of the patient fibroblasts with empty vector.After puromycin selection, cells were pelleted and sent to Sigma for deep sequencing. FANCA deficient patient fibroblasts (FANCA-/-) and their corrected counterparts (FANCA+) were plated (2 replicates/cell line) to 50% confluency in 15cm plates (Falcon). Cells were grown in DMEM supplemented with 10% FBS, 1% penn/strep, and 1% sodium pyruvate. All samples were transduced with the kinome-wide shRNA library. Cells were transduced per manufacturer's protocol with a lentiviral library (MISSION® LentiExpress™ Human Kinases, Sigma Aldrich) that encodes ∼5000 unique shRNA sequences targeting ∼516 human kinase genes, as well as non-targeting controls.  Cells were transduced at a multiplicity of infection (MOI) of ~0.5. A non-transduced plate served as a negative control. On day 3, cells were selected with 1 µg/ml puromycin media for 5 days.  Cells were treated with 1nM mitomycin c after shRNA transduction. Cells were treated with 1nM taxol after shRNA transduction. DNA extraction was performed by Sigma Aldrich. Sigma Aldrich provided the library.