GSM5627455: Pcgf4-/-Pcgf2flfl_ESC_TAM_rep1; Drosophila melanogaster; Mus musculus; RNA-Seq
2 x 10^7 mouse ESCs were mixed with 8 x 10^6 Drosophila SG4 cells in PBS. Nuclei were isolated 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. They were then recovered by centrifugation at 1000 × g for 5 min at 4°C, followed by a total of three washes with ice-cold RSB buffer (10 mM NaCl, 10 mM Tris (pH 8.0), 3 mM MgCl2). Nuclei integrity was assessed using 0.4% Trypan Blue staining (ThermoScientific). Next, nuclei were resuspended in 1 ml of TRIzol reagent (ThermoScientific) and RNA was extracted according to the manufacturer's protocol, followed by treatment with the TURBO DNA-free Kit (ThermoScientific). Quality of RNA was assessed using 2100 Bioanalyzer RNA 6000 Pico kit (Agilent) and high-quality RNA samples were depleted of rRNA using the NEBNext rRNA Depletion kit (NEB). RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA-seq kit (NEB) following manufacturer's guidelines. RNA-seq were sequenced as 80 bp (Med13/13lflfl) or 40 bp (Pcgf4-/-Pcgf2flfl) paired-end reads on the Illumina NextSeq 500 platform in biological quadruplicates