title: GSM2454410: mESC_nucRNA-Seq_TAM_rep3; Mus musculus; RNA-Seq
library_construction_protocol: 4 × 10^6 cells were resuspended in 1 mL HS Lysis buffer (50 mM KCl, 10 mM MgSO4.7H20, 5 mM HEPES, 0.05% NP40 (IGEPAL CA630)), 1 mM PMSF, 3 mM DTT)  for 1 min at room temperature to isolate nuclei. Nuclei were centrifuged at 1000 × g for 5 min at 4°C, followed by a total of three washes with ice-cold RSB buffer. Nuclei were then resuspended in 1mL of TriZOL reagent (ThermoScientific) according to the manufacturer’s protocol. Nuclear RNA was treated with the TURBO DNA-free Kit (ThermoScientific) and depleted for rRNA using the NEBNext rRNA Depletion Kit (NEB).   Nascent RNA sequencing was performed by pulse labelling with 4-thiouridine (4sU) as previously described (Rädle B. et al., 2013). Confluent 15 cm plates were treated with 500 µM 4sU in 10 ml culture medium for 20 min. The 4sU-containing media was then aspirated and immediately replaced with 5 ml TRIZOL (Thermo Fisher Scientific). RNA was isolated by phenol-chloroform extraction using phase lock tubes (Eppendorf) and resuspend in nuclease-free water. Total RNA aliquots were treated with 6U Turbo DNase (Ambion) and incubated at 37°C for 30 min, followed by inactivation of the DNase according to the manufacturer’s instructions. 300 μg of the resulting total RNA were incubated with 2 μg Biotin-HPDP/μg RNA in biotinylation buffer (10 mM Tris-HCl pH 7.4 and 1 mM EDTA) under rotation for 1.5 h at RT. Unincorporated biotin-HPDP was removed by chloroform/isoamylalcohol extraction. To capture biotinylated RNA, μMACS streptavidin beads (Miltenyi) were added to the RNA suspension (1 μl of beads/ μg RNA) and rotated for 15 min at room temperature prior to loading onto μMACS minicolumns. Unlabelled RNA was removed by washing three times with 900 µl of wash buffer (100 mM Tris-HCl pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween20) pre-heated at 65°C, then three more times with wash buffer at room temperature. Biotinylated RNA was then eluted in two rounds with 100 mM DTT and purified using an RNeasy miniElute kit (Qiagen). RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA-seq kit (NEB), whereas gDNA libraries were prepared using NEBNext Ultra DNA Library Prep Kit (NEB). Libraries were sequenced on the Illumina NextSeq500 with 80bp paired-end reads.