GSM3379446: PCGF3/5fl_UNT_NucRNA_rep2; Drosophila melanogaster; Mus musculus; RNA-Seq
1 x 10^7 mouse ESCs were mixed with 4 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). To quantitate the consistency of spike-in cell mixing for each individual sample, a small aliquot of nuclei was used to isolate genomic DNA using phenol-chloroform extraction. This was followed by sonication of DNA for 13-15 min using a BioRuptor Pico sonicator (Diagenode), shearing genomic DNA to an average size of less than 1 kb. RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA-seq kit (NEB) following manufacturer's guidelines. Libraries from sonicated genomic DNA were constructed using NEBNext Ultra DNA Library Prep Kit for Illumina and indexing was done using NEBNext Multiplex Oligos, following manufacturer's guidelines. Both RNA-seq and gDNA-seq libraries were sequenced as 80 bp paired-end reads on the Illumina NextSeq 500 platform in biological triplicate.
GEO Accession
GSM3379446