GSM7519193: T47D_Clone28_1hour_rep3; Homo sapiens; OTHER
Cell permeabilization was performed as previously described (Mahat et al. 2016), with modifications. At the time of harvest, cells were scraped in 10mL ice cold PBS and washed in 5mL buffer W (10mM Tris-HCl pH 7.5, 10mM KCl, 150mM sucrose, 5mM MgCl2, 0.5mM CaCl2, 0.5mM DTT, 0.004U/mL SUPERaseIN RNase inhibitor (Invitrogen), Complete protease inhibitors (Roche)). Cells were permeabilized by incubating with buffer P (10 mM Tris-HCl pH 7.5, KCl 10 mM, 250 mM sucrose , 5 mM MgCl2, 1 mM EGTA, 0.05% Tween-20, 0.1% NP40, 0.5 mM DTT, 0.004 units/mL SUPERaseIN RNase inhibitor (Invitrogen), Complete protease inhibitors (Roche)) for 3 minutes on ice. Cells were washed with 10 mL buffer W before being transferred into 1.5mL tubes using wide bore pipette tips. Finally, cells were resuspended in 50μL buffer F (50mM Tris-HCl pH 8, 5mM MgCl2, 0.1mM EDTA, 50% Glycerol, 0.5 mM DTT). Cells were snap frozen in liquid nitrogen and stored at -80°C. PRO-seq libraries were prepared as previously described (Judd et al. 2020), with modifications. RNA extraction after the run-on reaction was performed with 500μL Trizol LS (Thermo Fisher) followed by 130μL chloroform (Sigma). The equivalent of 1μL of 50μM for each adapter was used. A random eight base unique molecular identifier (UMI) was included at the 5′ end of the adapter ligated to the 3′ end of the nascent RNA. 37°C incubations were performed with rotation with 1.5mL tubes placed in 50mL conical tubes in a hybridization oven. For the reverse transcription reac- tion, RP1 was used at 100μM and dNTP mix was used at 10mM each. Libraries were amplified by PCR for a total of 8 cycles in 100μL reactions with Phusion polymerase (New England Biolabs). No PAGE purification was performed to ensure that our libraries were not biased against short nascent RNA insertions.