GSM5065647: Luc KD_GRO-seq_E2 rep2; Homo sapiens; OTHER
MCF-7 cells were washed three times with ice-cold PBS and then resuspended for swelling in ice-cold Hypotonic Lysis Buffer [10 mM Tris•HCl, pH 7.4, 0.5% NP-40, 10% Glycerol, 3 mM CaCl2, 2 mM MgCl2, and 1 mM DTT containing 1x protease inhibitor cocktail (Sigma-Aldrich) and 4 units/mL SUPERase-In (Ambion)]. The swollen cells were collected by centrifugation at 1000 RCF for 10 min at 4°C and then resuspended in 1.5 ml of lysis buffer and pipetted up and down through a narrow opening tip 30 to 50 times to lyse the cells and release the nuclei. The nuclei were collected by centrifugation and washed once with 1 mL of Hypotonic Lysis Buffer. After a final collection by centrifugation, the resulting pellets of nuclei were resuspended in 500 µL of Freezing Buffer (50 mM Tris•HCl, pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, and 4 units/mL of SUPERase-In per mL), counted, frozen in liquid nitrogen in 100 µL aliquots containing 5 x 106 nuclei, and stored at -80°C until use. (PMID: 7256882) GRO-seq libraries were prepared as described previously with some minor modifications (PMID: 24564208).