Cell permeablization and PRO-seq

Samples were then prepared for precision run-on reactions by subjecting cells to permeablizing conditions. Briefly, cultures were spun down and resuspended in ice cold 1×PBS. Samples were spun again and washed in 5 mL wash buffer (10 mM Tris-Cl, pH 7.5, 10 mM KCl, 150 mM sucrose, 5 mM MgCl₂, 0.5 mM CaCl₂, 0.5 mM DTT 1× protease inhibitor cocktail [Roche], and 20 U RNase inhibitor [SUPERase In, Invitrogen]). Cell pellets were then resuspended in permeabilization buffer (10 mM Tris-Cl, pH 7.5, 10 mM KCl, 250 mM sucrose, 5 mM MgCl₂, 1 mM EGTA, 0.05% Tween-20, 0.1% NP40, 0.5 mM DTT, 1× protease inhibitor cocktail [Roche], and 20 U RNase inhibitor [SUPERase In, Invitrogen]) and left on ice for 5 min. Cells were checked for penetration by trypan blue to assess permeability (∼99% permeable). Cells were then washed two times in 5 mL wash buffer before being resuspended in 200 µL storage buffer (50mM Tris-Cl, pH 8.3, 40% glycerol, 5 mM MgCl₂, 0.1 mM EDTA, and 0.5 mM DTT). A 1:50 dilution was prepared using 2 µL of each sample and used to take OD₆₀₀ measurements. All samples were then diluted to an equal density (OD₆₀₀ = 0.181) in a final volume of 110 µL of storage buffer. Then, 5 × 10⁴ pre-permeabilized S2 cells were spiked in to each cell count-normalized sample before flash-freezing the permeabilized cells and storing them at −80°C.

Stored permeable cells with spike-ins were thawed on ice, and each sample was subjected to the precision run-on protocol (Mahat et al. 2016a). Run-on reactions incorporated only biotinylated NTPs with no unmodified NTPs. All libraries were subjected to nine cycles of PCR amplification before size selection and gel purification.