title: GSM9103346: 4sU_RNA_gTOP2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA (200-300 µg) was used for biotinylation and purification of 4sU-labeled nascent RNAs. 1 µg spike-in from yeast per 100 µg total RNA was added. Total RNA was fragmentated using Bioruptor Pico. The biotynilation reaction consisted of total fragmented RNA and EZLink HPDP-Biotin (Thermo Scientific, 20217) dissolved in dimethylformamide (DMF) and was performed in labeling buffer (10 mM Tris pH 7.4, 1 mM EDTA) for 2h with rotation at room temperature. Unbound Biotin-HPDP was removed by chloroform extraction. RNA was precipitated with 10th volume of 5M NaCl and 1 volume of isopropanol. Following one wash in 75% ethanol, the RNA pellet was left to dry and resuspended in 100 µl RNase-free water. Biotinylated RNA was purified using µMACs Streptavidin kit (Miltenyi Biotec, 130-074-101). Specifically, 100 µl of beads per 100 µg of RNA was incubated with rotation for 15 min and then washed three times with washing buffer (100 mM Tris pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween-20) at 65ºC, followed by three washes at room temperature. RNA was eluted twice using 100 mM DTT (Sigma) and recovered using miRNeasy Micro Kit (Qiagen, 217084) according to instructions. Libraries preparation of chromatin-associated RNA were done using Kapa RNA HyperPrep (Roche)