title: GSM2612360: K1-CDA-ER 4sU-RNA-seq rep2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted using TRIzol® Reagent (Life Technologies, #15596026) according to the manufacturer’s instructions with manually dissociated cells washed in 1 ml DPBS (Gibco™, #14190-136). Extracted RNA was subsequently treated with DNase I (New England Biolabs, #M0303L) to remove residual gDNA contamination. Isolation of 4sU-RNA was performed essentially as previously described (Rabani et al. Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells. Nat Biotechnol 2011) with minor modifications. Purified RNA was biotinylated with EZ-Link Biotin-HPDP (Pierce; #21341) and isolated using µMacs Streptavidin Kit (Miltenyi; #130-074-101). 4sU-enriched RNA was then purified with RNeasy MinElute Spin Columns (Qiagen; #74204). Purified 4sU-labelled RNA was used with the Ion Total RNA-Seq Kit v2 (Life Technologies; #4479789) to prepare libraries for sequencing on the Ion Proton™ System.