To collect interphase cells, media containing translation inhibitor was poured into a 50mL tube, cells were washed with PBS + 100 μg/mL cycloheximide and then trypsinized in the presence of 100 μg/mL cycloheximide for 3 minutes at 37°C. Cells were then collected using the cold media with inhibitor. For mitotic cells, after addition of cycloheximide, mitotic cells were harvested by shake off and incubated on ice. Cells were centrifuged at 500x g for 3 minutes, washed once with 20 mL PBS+cycloheximide, and the pellet was moved into a new 1.5 mL tube with 1 mL PBS+cycloheximide. Cells were lysed in 900 μL polysome lysis buffer (20 mM Tris pH 7.4, 100 mM KCl, 5 mM MgCl2, 1% (v/v) Triton X-100, 100 μg/mL cycloheximide, 500 U/mL RNaseIn Plus, 1x cOmplete protease inhibitor cocktail) and incubated on ice for 10 minutes. To ensure efficient lysis, cells were passed through a syringe 5 times. Cell debris and nucleus was depleted by spinning the lysate at 1300x g for 10 minutes. 30 μL of the cleared lysate was added to 400 μL TRI Reagent (Invitrogen, AM9738) for input RNA sequencing library. Three 300μL aliquots were generated with the rest of the cleared lysate, flash frozen in liquid nitrogen, and stored at -80°C.