Transfection of DNA library into 293T and PC3 cell lines

A total of 2.6 × 10⁶ 293T cells were plated onto a 15 cm dish, incubated overnight, and transfected with 16 µg of plasmid DNA library using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s protocol. At 24 h after transfection, cells were washed with phosphate-buffered saline (PBS), harvested with 0.05% Trypsin-EDTA (Gibco), and centrifuged at 300 × g for 5 min into a cell pellet. For the PC3 cell line, 3 × 10⁶ cells were plated onto a 15 cm dish and transfected with 16 µg of plasmid DNA library using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s protocol. At 24 h after transfection, cells were washed with PBS, harvested with 0.05% Trypsin-EDTA (Gibco), and centrifuged at 300 × g for 5 min into a cell pellet. A total of 2.6 × 10⁶–3 × 10⁶ cells/plate were chosen to enable over 900× coverage of the plasmid library per replicate (assuming 100 plasmids/cell, >25% transfection efficiency, and 212,325 unique constructs within the library). In both cell lines, cell pellets collected from each 15 cm dish were resuspended in 1 mL of cold PBS (Gibco) + 100 µg cycloheximide (Sigma) and incubated on ice for 10 min. The cells were centrifuged into a cell pellet and lysed in 220 µL of lysis buffer (Tris-HCl, NaCl, MgCl₂, 10% NP-40, Triton X-100, SUPERase In RNase Inhibitor, cycloheximide, dithiothreitol, diethyl pyrocarbonate water) for 45 min on ice, and vortexed every 10 min. For each cell line, lysates from three 15 cm dishes were pooled together to form one biological replicate. A total of three biological replicates were performed for each cell line. From each replicate, 60 µL of lysate was collected for DNA extraction using the QIAprep Spin Miniprep Kit (Qiagen). To collect total mRNA, 800 µL of Trizol (Life Technologies) was added to 150 µL of lysate and stored at −80 °C.