title: GSM9232928: RPB1degron_32nM_45min_DRB_release_1; Homo sapiens; OTHER
library_construction_protocol: Following a 10 minute pulse with 1mM 4sU, media was aspirated and cells were harvested by the addition of 1mL Trizol to stop the reaction. To isolate total RNA, one-fifth of chloroform was added to lysates and shaken well, followed by centrifugation (12000g, 5 minutes at 4C). The upper aqueous phase was transferred to MaXtract high-density phase-lock tubes, that were previously prepared according to manufacturer's instructions, and an equal volume of chloroform/isoamyl alcohol (24:1) was added. Lysates were then centrifuged again (12000g, 5 minutes at 4C) and the upper aqueous phase was transferred to a new tube. The RNA was then precipitated through the addition of 1.1X volumes of 100% isopropanol, incubation at room temperature for 20 minutes and centrifugation (12000g, 20 minutes at 4C). RNA pellets were washed once with 85% (v/v) ethanol, and air dried at room temperature before resuspension in Ultrapure DNase/RNase-free distilled water. As an external reference control, 500ng S.cerevisiae (strain BY4741, MATa, his3D1, leu2D0, met15D0, ura3D0) nascent RNA was spiked-in to 100µg of H.sapiens total RNA. The RNA was then fragmented by the addition of 20µL 1M NaOH and incubation on ice for 20 minutes. Fragmentation was stopped by the addition of 80µL 1M Tris HCl pH 6.8 and material was purified twice using the Micro Bio-Spin P-30 Gel Columns according to manufacturer's instructions. Thiol-specific biotinylation of RNA was performed using 10 mM Tris-HCl pH 7.4, 1 mM EDTA and 5 mg MTSEA biotin-XX linker in a final volume of 250µL and incubation at room temperature for 30 minutes in the dark. Biotinylated RNA was then purified by addition of 250μL of phenol/chloroform/isoamyl alcohol (25:24:1 (vol/vol)), followed by shaking and addition to MaXtract high-density phase-lock tubes that were previously prepared according to manufacturers instructions. Following centrifugation (12000g, 5minutes at 4C), the upper aqueous phase was transferred to a new tube and biotinylated RNA was precipitated using one-tenth the volume 5M NaCl and 1.1X volume 100% isopropanol, followed by incubation at room temperature for 10 minutes and centrifugation (20000g, 20 minutes at 4C). RNA pellets were washed once in 85% (v/v) ethanol, air dried at room temperature, and resuspended in 50µL Ultrapure DNase/RNase-free distilled water. The RNA was then denatured by incubating for 10 minutes at 65°C in the dark. Biotinylated RNA was separated from the total RNA pool by addition of 100 μL μMACS Streptavidin MicroBeads and incubation for 15 minutes at room temperature with constant rotation. Beads were then applied to a μColumn pre-equilibrated with 100 µL nucleic acid equilibration buffer and immobilised on a magnetic stand, washed twice with pre-warmed (55°C) wash buffer (100 mM Tris HCl pH 7.4 10mM EDTA, 1M NaCl and 0.1% (v/v) Tween 20) and biotinylated RNA was eluted by two additions of 100μL 100 mM DTT, 5 minutes apart. Purification of biotinylated RNA was performed using the RNeasy MinElute kit according to manufacturer's instructions, where 1050 μL 100% ethanol and 750 μL RLT buffer was added per 200μL reaction to precipitate RNA < 200 nt. Sequencing libraries were prepared from biotinylated RNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (without additional fragmentation) and NEBNextMultiplex Oligos for Illumina (Unique Dual Index UMI adapters RNA Set 1) and were sequenced on the NextSeq 2000 (P3 50 kit).