title: GSM4829014: RING1AKO.RING1BAID_NucRNA_UNT_rep2; Drosophila melanogaster; Mus musculus; RNA-Seq
library_construction_protocol: 2x107 mouse ESCs (untreated or treated with auxin for indicated times) were mixed with 8x106 Drosophila SG4 cells in PBS. Nuclei were released in 1 mL HS Lysis buffer (50 mM KCl, 10 mM MgSO4.7H20, 5 mM HEPES, 0.05% NP40, 1 mM PMSF, 3 mM DTT, 1x PIC) for 1 min at room temperature, and recovered by centrifugation at 1000 g for 5 min at 4°C, followed by three washes with ice-cold RSB buffer (10 mM NaCl, 10 mM Tris pH 7.4, 3 mM MgCl2). Pelleted nuclei were resuspended in 1 mL of TRIzol reagent and RNA was extracted according to the manufacturer's protocol, followed by treatment with the TURBO DNA-free Kit. Quality of RNA was assessed using 2100 Bioanalyzer RNA 6000 Pico kit (Agilent), followed by rRNA depletion using the NEBNext rRNA Depletion kit (NEB). RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA Library Prep kit (NEB), indexed using NEBNext Multiplex Oligos (NEB), polled and sequenced as 80 bp paired-end reads on the Illumina NextSeq 500 in biological triplicates.