title: GSM5258083: SUDHL1_RG1_PROseq; Homo sapiens; OTHER
library_construction_protocol: Cells were washed once in ice-cold 1x PBS and resuspended in Wash Buffer at the density of 5x10^6 cells/mL. Once filtered to ensure single-cell suspension, 9x volume of Permeabilization Buffer (10mM Tris-HCl pH 8, 10mM KCl, 250mM Sucrose, 5mM MgCl2, 1mM EDTA, 0.5mM DTT, 10% Glycerol, 0.05% Igepal CA-630) was gently added. The cells were incubated on ice for 3 minutes and then recovered by centrifugation and washed in Freezing Buffer (50mM Tris-HCl pH 8.3, 40% Glycerol, 5mM MgCl2. 0.5mM DTT, 2µL/mL RNase Inhibitor). Permeabilized cells were resuspended in Freezing Buffer at a density of 1x10^6 cells/100µL, flash frozen, and stored at -80°C until library preparation. PRO-seq libraries were generated as previously described (DOI: 10.1016/j.molcel.2019.10.034).