GSM8253867: PRO_dTAG_Serum10_Replicate1; Homo sapiens; OTHER
Cells were processed for PRO-Seq by washing and permeabilization as described in the methods of the manuscript. The qPRO-Seq protocol was followed with minor modifications (Judd et al. 2020). Cells were seeded in 10 cm tissue culture dishes, treated as indicated and were ~90% confluent prior to collection. For each PRO-Seq, ~4E6 cells were utilized. To prepare permeabilized cells for run-on, cells were first washed twice on the plate with 5 mL of ice-cold 1X PBS. Then, 2.5 mL of ice-cold Cell Permeabilization Buffer (CPB) (10 mM Tris-Cl pH = 8.0 , 250 mM Sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA pH = 8.0, 0.1% NP-40, 0.5 mM DTT, 0.05% Tween-20, 0.1% Triton X-100, 10% Glycerol, 1X protease inhibitor, and 2 µL SUPERase-In RNase inhibitor (Thermo, catalog AM2696) per 10 mL was immediately added to the tissue culture dish. Triton X-100 was added to CPB to increase permeabilization percent. Dishes were placed on ice and then immediately scraped with a cell scraper. At this point, cells were collected in a 15 mL tube, placed on ice for 5 min, checked for permeabilization by trypan blue staining (>95%), and then centrifuged in a swinging bucket rotor (1000xg, 4 min, 4°C). Following this, cells were handled using cut tips. The supernatant was removed and samples were resuspended in 1 mL of Cell Wash Buffer (CWB) (10 mM Tris-Cl pH = 8.0, 250 mM Sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA pH = 8.0, 0.5 mM DTT, 10% Glycerol, 1X protease inhibitor, and 2 µL SUPERase-In RNase inhibitor per 10 mL) and centrifuged (1000xg, 4 min, 4°C) for a total of 2 washes. After the second wash, samples were resuspended in 1 mL total Cell Freeze Buffer (CFB) (50 mM Tris-Cl pH = 8.0, 5 mM MgCl2, 0.5 mM DTT, 40% Glycerol, 1.1 mM EDTA pH = 8.0, and 2 µL SUPERase-In RNase inhibitor per 10 mL), counted by hemacytometer, and then spun down in 1.5 mL tubes in an angled rotor centrifuge (1000xg, 5 min, 4°C). Samples were resuspended in 52 μL of CFB for every 4E6 cells, flash frozen in liquid nitrogen, and then frozen at -80°C until ready to use. Library was prepared with individual reagents following the protocol from Judd et al (dx.doi.org/10.17504/protocols.io.57dg9i6) exactly as described.