GSM9214929: D21 untreated RNA-seq, rep3; Homo sapiens; RNA-Seq
For PRO-seq, nuclei were isolated as follows: Post-treatment, cells were placed on ice and washed three times with ice-cold PBS. Cells were incubated on ice in 10 ml ice-cold Lysis Buffer (10 mM Tris-HCl pH 7.5, 2 mM MgCl2, 3 mM CaCl2, 0.5% IGEPAL, 10% Glycerol, 2 U/ml SUPERase-IN, brought to volume with 0.1% DEPC DI-water, filtered before use) for 10 minutes. Cells were scraped and collected into 50 ml Falcon tubes, and centrifuged with a fixed-angle rotor at 1000 x g for 10 minutes at 4 C. Cells were resuspended with Lysis buffer with a wide-opening P1000 tip, and washed twice with 10 ml Lysis buffer (centrifuged at 1000 x g for 5 minutes at 4 C). After the second Lysis buffer wash, the samples were resuspended with 1 ml Freezing Buffer (50 mM Tris-HCl pH 8.3, 5 mM MgCl2, 40% Glycerol, 0.1 mM EDTA pH 8.0, brought to volume with 0.1% DEPC DI-water, filtered before use). Nuclei were centrifuged at 1000 x g for 5 minutes at 4 C, and resuspended with 500 ul Freezing Buffer. Nuclei were then centrifuged for 2 minutes at 2000 x g, 4 C, and resuspended in 110 ul Freezing Buffer. Sample was snap-frozen in liquid nitrogen and stored at -80 C until use. For RNA-seq, cells were first pelleted and washed twice with ice-cold PBS. The pellet was dissolved with 500 ul trizol, and RNA chloroform-extracted with 130 ul chloroform For PRO-seq, libraries were constructed per Mahat et al, 2016. For RNA-seq, libraries were constructed using KAPA RNA Hyperprep Kit with RiboErase