GSM8478231: G1E-ER4, Control, Replicate2; Mus musculus; RNA-Seq
Cell pellets were lysed in 1 mL of TRIzol™️ Reagent (Invitrogen). After a 5-minute incubation at room temperature, 200 μL of RNase-free chloroform (Sigma-Aldrich) was added followed by vigorous mixing. The samples were further incubated for 15 minutes at room temperature and then centrifuged at 14,000 g for 15 minutes at 4°C to separate the phases. The uppermost aqueous layer was carefully transferred to sterile 1.5 mL Eppendorf tubes, and 500 μL of isopropanol (Sigma-Aldrich) and 1 μL of glycogen (Sigma-Aldrich) were added and gently mixed by inversion. The RNA samples were then incubated overnight at -20°C. On the next day, the samples were thawed and centrifuged at 12,000 g for 20 minutes at 4°C. The supernatant was discarded, and the RNA pellet was washed with 1 mL of 75% (v/v) ethanol. After further centrifugation at 12,000 g for 5 minutes at 4°C, the supernatant was removed, and the RNA pellet was allowed to air-dry. Finally, 30 μL of RNase-free, UltraPureTM distilled water (Gibco) was added, and the RNA samples were stored at -80°C. Total RNA (1-3μg/sample) extracted were submitted to the Australian Genome Research Facility Core Facility for quality control analysis. The RNA quality was assessed using the Agilent 2100 Bioanalyzer, and the RNA integrity number for all samples measured >9. The poly(A)-RNA was captured using oligo-dT beads and used for cDNA library preparation using the standard TruSeq RNA Sample Prep Kit v2 protocol (Illumina, CA, USA). Briefly, total RNA was poly-A selected and then fragmented. The cDNA was synthesized using random hexamers, end-repaired and ligated with appropriate adaptors for sequencing. The library then underwent size selection and purification using AMPure XP beads (Beckman Coulter, CA, USA). The appropriate Illumina recommended 6 bp barcode bases are introduced at one end of the adaptors during PCR amplification step. The size and concentration of the RNAseq libraried was measured by Bioanalyzer and Oubit fluorometry (Life Technologies, NY, USA) before loading onto the sequencer. The mRNA libraries were sequenced on the Illumina HiSeq 2000 System with 100 nucleotide paired-end reads, according to the standard manufacturer's protocol (Illumina, CA, USA).