GSM8146837: HCT116, DMSO 30min 1; Homo sapiens; OTHER
At time 30 minutes, the media was aspirated from the plate and the cells were washed in ice cold 1x D-PBS three times. 40mL of ice-cold Lysis Buffer (10 mM Tris–HCl pH 7.4, 2 mM MgCl2, 3 mM CaCl2, 0.5% NP-40, 10% glycerol, 1 mM DTT, 1x Protease Inhibitors (1mM Benzamidine (Sigma B6506-100G), 1mM Sodium Metabisulfite (Sigma 255556-100G), 0.25mM Phenylmethylsulfonyl Fluoride (American Bioanalytical AB01620), and 4U/mL SUPERase-In) was then added to the plate and cells were scraped with a cell-lifter (Costar) and the resuspended cells were transferred into a conical and put on ice. Resuspended cells were allowed to swell in lysis buffer, on ice, for 0.5-1hour (while the other cells were being harvested). Cells were then pelleted in a fixed-angle rotor centrifuge at 1000xg for 15 minutes. Pelleted cells were resuspended in 10mL of Lysis Buffer with a wide-bore P1000 tip and the centrifugation step was repeated. Being mostly nuclei and cell debris, the pellet was resuspended in 500uL of Freezing Buffer (50 mM Tris pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 4U/ml SUPERase-In) and moved to a low-retention 1.5mL tube and pelleted at 2000xg for 4 minutes. The final pellet was resuspended in Freezing Buffer, checked for purity and counted under a microscope, and brought to a concentration of 10million nuclei/100uL then aliquoted and snap frozen. Nuclear run-on was conducted as described (Mahat et al., 2016) with the following modifications: the final concentration of non-biotinylated CTP was raised from 0.25mM to 25mM, and the final library clean-up and size selection was accomplished using 1X AMPure XP beads (Beckman A63881).