14-Rep2-dox, 37
A549cHSF1(p53-Lib) cells were seeded in 15 cm tissue culture plates at a density of 3  106 cells/plate. In order to maintain library diversity throughout selection, three plates were used per treatment for a total of 9  106 cells. Cells were treated with 0.01% DMSO, 1 g/mL dox, 0.01% DMSO and 2.5 M nutlin-3, or 1 g/mL dox and 2.5 M nutlin-3. Cells were trypsinized, counted, and re-seeded in three plates each at 3  106 cells/plate every 3 d. Following 12 d of treatment, cell pellets were harvested by centrifugation at 1,000 rpm for 5 min. Aliquots of 9  106 cells were snap-frozen in liquid N2(g) in Eppendorf tubes and stored at 80 C for subsequent DNA extraction. The deep mutational scanning experiment was repeated independently for a total of three biological replicates from the same p53-Lib cell line. Genomic DNA was purified from aliquots of frozen cells using the QIAamp Blood Midi Kit (Qiagen) and final DNA concentration was determined using a Qubit (Fischer). PCR amplicons of p53 were prepared using 2.0 g of genomic DNA over 25 cycles and with Herculase II as the DNA polymerase (Agilent). The primers used were 5' ATTCTCCTTGGAATTTGCCCTT 3' and 5' CATAGCGTAAAAGGAGCAACA 3'. Twelve PCR reactions were performed per sample, and the reactions were pooled and cleaned up using a PCR clean-up kit (Omega). The p53 amplicons were further gel-purified using a Pippin prep system (Sage Science) prior to library preparation via Nextera Flex.
SUB14814136
D21-4386-5116R