GSM8172189: ctr1; Homo sapiens; RNA-Seq
Total RNA was isolated from cells using TRIzol reagent (Ambion). About 1 million cells were lysed in 1 mL of TRIzol followed by the addition of 0.2 mL (5:1 ratio) of chloroform. Samples were then centrifuged at 12,000 g for 15 min at 4℃. The top layer was transferred to a tube and the RNA was precipitated in 100% isopropanol (1:1 ratio), 3 M sodium acetate (10:1 ratio), and 4 μL of glycogen (5 mg/mL) for an hour at -20℃. The samples were then centrifuged at 15,000 RPM for 20 min at 4℃ and washed twice with 1 mL of 75% ethanol. RNA was isolated as described above. About 1 μg of total RNA was sent to Admera Health for library construction. The NEBNext Poly(A) mRNA Magnetic Isolation Module was used to isolate intact poly(A)+ RNA followed by NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®. Library concentration was measured by qPCR, and library quality was evaluated by Tapestation High Sensitivity D1000 ScreenTapes (Agilent Technologies, CA, USA). Equimolar pooling of libraries was performed based on qPCR values.