GSM7430017: Hela, 90min, nucleus, rep1; Homo sapiens; OTHER
45 million cells were used per sample. The medium was supplemented with 500 μM for either 0, 15, 30, 45, 60, 90, 120 or 180 min immediately  followed by subcellular fractionation as described in Nojima et al. (2016) with modifications as follows: the cells were rinsed on the cell culture plates twice with 20 ml of ice-cold DPBS twice and scraped in 10ml ice-cold DPBS and transfered into a 15-ml tube. The cells were pelleted at 400 x g or 5 min at 4°C, resuspended in 4 ml of ice-cold HLB+N buffer (10mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2 and 0.5% (vol/vol) NP-40) followed by an incubation on ice for 5 min. The cell pellet was underlaid with 1 ml of ice-cold HLB+NS buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2, 0.5 % (vol/vol) NP-40 and 10 % (wt/vol) sucrose). The nuclei were pelleted by centrifugation at 400 x g for 5 min at 4 °C. The supernatant was the collected and lysed with 3 volumes of Trizol LS for cytoplasmic fraction. The nuclei were washed with 15 ml ice-cold DPBS and lysed in Qiazol. For the total fractions we lysed the metabolically labeled cells directly in Qiazol. Synthetic spike-ins were produced and purified. The preparation protocol was modified to include in vitro poly(A) tailing with E. coli Poly(A) Polymerase (NEB) to make the spike-ins compatible with the NGS library preparation. We added equal amounts of synthetic spike-ins to all the RNA fraction lysates prior to RNA isolation. RNA was isolated according to the manufacturer's protocol and then dissolved in nuclease-free water with 1 mM DTT. The carboxyamidomethylation reactions of 4sU were set up as described by Herzog et al. Briefly described here, we set up the reaction with 5 μg RNA in nuclease-free water with 1 mM DTT; 50% DMSO; 50 mM sodium phosphate buffer pH 8.0; and 10 mM iodoacetamide. The reactions were incubated at 50°C for 15 min and then quenched by 100 mM DTT. The RNA was then precipitated with ethanol to clean up and concentrate. Next generation sequencing libraries were prepared from 500 ng RNA with the QuantSeq 3' mRNA-Seq Library Prep Kit REV for Illumina according to the manufacturer's instructions, but with two modifications. In brief, the reverse transcription reaction temperature was increased to 50°C and the incubation was extended to 60 min. The NGS libraries were pooled and sequenced as single-read with the custom sequencing primer provided with the QuantSeq NGS library preparation kit with 100 cycles on a HiSeq2000 sequencer.