GSM8900943: U87, cytoplasmic fraction, DMSO-treated, Rep 2; Homo sapiens; RNA-Seq
For whole cell lysates, following 3-day treatment with either DMSO (vehicle), PRMT5i, or dTag-13, cells were harvested by scraping. RNA was purified using the RNeasy Mini Kit (Qiagen, 74106) according to manufacturer's instructions, including the optional DNaseI addition to remove contaminating gDNA (Qiagen, 79254). For fractionated cells, after 3 days in drug, cells were harvested via trypsinization, pelleted, and separated into nuclear and cytoplasmic fractions. Briefly, cell pellets were resuspended in cytoplasmic lysis buffer [0.15% (v/v) NP40, 10 mM Tris (pH7), 150 mM NaCl] supplemented with 10 U SUPERase.IN (Thermo Fisher, AM2694) and EDTA-free protease inhibitor cocktail (MilliporeSigma, 11836170001). Cells were then incubated on ice for 5 minutes. Cell lysate was layered on top of sucrose buffer [0 mM Tris (pH7), 150 mM NaCl, 25% (w/v) sucrose] supplemented with 10 U SUPERase.IN (Thermo Fisher, AM2694) and EDTA-free protease inhibitor cocktail (MilliporeSigma, 11836170001). Cells were then centrifuged at 16,000 x g for 10 minutes at 4 °C. The supernatant contains the cytoplasmic fraction and was harvested for RNA extraction. The remaining pellet contains the nuclear fraction and was washed twice with 800 μL cold PBS, centrifuging at 16,000 x g for 1 minute at 4°C after each wash. The pellet was resuspended in cold RIPA buffer [50 mM Tris (pH 8), 150 mM NaCl, 1% (v/v) NP-40, 0.5% sodium deoxycholate, and 0.1% (w/v) SDS] supplemented with 10 U SUPERase.IN (ThermoFisher, AM2694) and EDTA-free protease inhibitor cocktail (MilliporeSigma, 11836170001). Lysate was incubated for 30 minutes on ice, with vortexing every 10 minutes, and then pelleted at 16,000 x g for 10 minutes at 4°C. The supernatant contains the nuclear fraction and was harvested for RNA extraction. The RNA from the nuclear and cytoplasmic fractions were extracted using TRIzol Reagent (Thermo Fisher, 15596026) according to manufacturer's instructions. Briefly, to each aqueous fraction, 0.5 mL of isopropanol and 1 mL of TRIzol Reagent was added, followed by an incubation on ice for 10 minutes. Samples were then centrifuged for 10 minutes at 12,000 x g at 4 °C, pellets were washed in 1 mL 75% ethanol, and centrifuged again for 5 minutes at 7,500xg at 4 °C. Pellets were air dried for 10 minutes and resuspended in RNase-free water. RNA-Seq libraries were prepared using the NEB Ultra II Directional RNA Kit with Poly(A) Selection per the manufacturer's protocol.