title: GSM4929479: PRC1CPM_NucRNAseq_OHT_rep2; Drosophila melanogaster; Mus musculus; RNA-Seq
library_construction_protocol: For cnRNA-seq, 1×10^7 ESCs (untreated and OHT-treated) were mixed with 4×10^6 Drosophila SG4 cells in 1x 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, 1xPIC (Roche)) for 1 min at room temperature, and 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, 3 mM MgCl2). Nuclei integrity was assessed using 0.4% Trypan Blue staining (ThermoScientific). Pelleted 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) to remove any contaminating DNA. Quality of RNA was assessed using the 2100 Bioanalyzer RNA 6000 Pico kit (Agilent). RNA samples were depleted of rRNA with the NEBNext rRNA Depletion kit (NEB) prior to library prep. To quantitate the consistency of spike-in cell mixing for each individual sample, a small aliquot of nuclei was saved to isolate genomic DNA using phenol-chloroform extraction. This was followed by sonication of DNA for 15 min using the BioRuptor Pico (Diagenode), shearing genomic DNA to an average size of less than 1 kb. cnRNA-seq libraries were prepared using the NEBNext Ultra (for Bap1fl/fl and PRC1CPM) or Ultra II (for PRC1CPM;Bap1fl/fl) Directional RNA Library Prep kit (NEB). Libraries from sonicated genomic DNA were constructed using NEBNext Ultra (for Bap1fl/fl and PRC1CPM) or Ultra II (for PRC1CPM;Bap1fl/fl) DNA Library Prep Kit for Illumina, following manufacturer's guidelines. Samples were indexed using NEBNext Multiplex Oligos. The average size and concentration of all libraries were analysed using the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent) followed by qPCR quantification using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche). Both cnRNA-seq and gDNA-seq libraries were sequenced as 80 bp paired-end reads on the Illumina NextSeq 500 platform in biological triplicates.
attributes.tag: GEO Accession
attributes.value: GSM4929479