GSM4458916: HEK293T rna-seq from N_2A15_2; Homo sapiens; RNA-Seq
Cell fractionation by sequential detergent extraction was performed as previously described (Jagannathan et al., 2011, doi: 10.1007/978-1-61779-005-8_19 ) with minor modifications. HEK293 and HEK293 HDLBP knockout cell lines (1 15 cm dish per replicate) were grown to ~90 % confluency. Cells were washed with PBS. All further steps were carried out on ice using ice cold reagents and cells were always pelleted at 3000 g for 5 min at 4°C. First, PBS containing 50 µg/ml cycloheximide was added for 10 min. In the meantime cells were scraped using a rubber policeman. After pelleting, the cells were resuspended with 500 µl permeabilization buffer (110 mM KOAc, 25 mM K-HEPES [pH 7. 2], 2.5 mM Mg(OAc)2, 1 mM EGTA, 0.015 % digitonin, 1 mM DTT, 50 µg/ml cycloheximide, complete EDTA-free protease inhibitor cocktail [Roche], 40 U/mL SUPERaseIn [Thermo Fisher Scientifc]) per sample and incubated for 15 min at 4°C on a rotating wheel. After centrifugation the supernatant was collected as the cytosolic fraction. To wash the pellet, 5 ml of washing buffer (110 mM KOAc, 25 mM K-HEPES [pH 7.2], 2.5 mM Mg(OAc)2, 1mM EGTA, 0.004% digitonin, 1 mM DTT, 50 μg/ml cycloheximide) was used. After pelleting, 500 µl lysis buffer (400 mM KOAc, 25 mM K-HEPES [pH 7.2], 15 mM Mg(OAc)2, 0.5 % IGEPAL CA-630, 1 mM DTT, 50 μg/ml cycloheximide, complete EDTA-free protease inhibitor cocktail, 40 U/mL SUPERase•In) per sample was added for 5 min. After centrifugation, the supernatant was collected as the membrane fraction. The remaining pellet  was resuspended with 500 µl lysis buffer, 200 µl 10 % sucrose in lysis buffer was added and the sample were centrifuged at 14000 rpm for 5 min at 4°C. The supernatant was removed, 200 µl wash buffer was added. After centrifucation the supernantant was remvoed and the pellet was collected as the nucleus fraction. Cytosol and membrane fractions were clarified by centrifugation at 7500 g for 10 min at 4°C. 250 µl of each sample was collected and RNA was isolated using Trizol LS (Thermo Fisher Scientific) in combination with RNA Clean & Concentrator-25 kit (Zymo Research) for RNA sequencing. Illumina TruSeq Stranded mRNA Kit