title: GSM7794510: 4sU shCTR9 Dox rep2; Mus musculus; OTHER
library_construction_protocol: Subconfluent cells were treated as described. During the last 15 min of treatment, cells were incubated with 2mM 4-thiouridine (4sU; Sigma-Aldrich) before lysis with QIAzol reagent (QIAGEN). Total RNA extraction was performed using the miRNeasy kit (QIAGEN) with on-column DNase digestion, and RNA quantity and quality were assessed using a Nanodrop spectrophotometer and a Fragment Analyzer (Thermo Fisher Scientific), respectively. 20-40 µg of RNA with RQN values greater than 9 was then biotinylated by adding EZ-Link Biotin-HPDP (Pierce) in 0.2 mg/ml dimethylformamide and biotin labeling buffer (10 mM Tris pH 7.4, 1 mM EDTA) and incubating for 2h at RT with rotation. Biotinylated RNA was then purified by chloroform isoamyl alcohol extraction in MaXtract high-density tubes (QIAGEN). The upper aqueous phase was collected and 1/10 volume of 5 M NaCl and an equal volume of isopropanol were added. All samples were centrifuged at 20,000g for 20 min at 4°C, the RNA pellets were washed with 75% ethanol and centrifuged at 20,000g for 10 min at 4°C. After drying, the pellets were resuspended in nuclease-free water. Biotinylated RNA samples were then eluted using Dynabeads MyOne Streptavidin T1 beads (Invitrogen) in Dynabeads Wash Buffer (2 M NaCl, 10 mM Tris pH 7.5, 1 mM EDTA, 0.1% v/v Tween 20) for 15 min at RT with rotation. After stringent washing, 4sU-labeled RNA was eluted from the beads with 100 ml of freshly prepared 100 mM DTT in nuclease-free water and purified using the RNeasy MinElute Kit (QIAGEN). Samples were quantified using the RiboGreen Assay (Invitrogen), and 5-20 ng RNA was used for cDNA library preparation by first depleting rRNA using the NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (New England Biolabs) and then the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs). cDNA libraries were amplified using appropriate multiplexed index primers in 11-14 cycle PCR, depending on the amount of RNA input.