GSM6856758: 72-SP-G2-1; Homo sapiens; RNA-Seq
Approximately 1 × 106 cells were used for RNA extraction. Cells were washed once with 1–2 mL pre-chilled PBS, resuspended in 1 mL TRIzol™, and incubated for 5 min at room temperature. Subsequently, 250 µL chloroform was added to the cell suspension, shaken vigorously for 15 s, and incubated for 5 min at room temperature. The cell suspension was centrifuged at 12,000 rpm for 15 min at 4°C, the aqueous phase was transferred to a clean 1.5-mL EP tube, 550 µL isopropanol was added and mixed gently, and the solution was incubated at room temperature for 5 min. The solution was centrifuged again at 4°C for 10 min at 12,000 rpm, the supernatant was discarded, and the RNA precipitate was resuspended in 1 mL pre-chilled 75% ethanol. The solution was centrifuged again at 7,500 rpm for 5 min, the supernatant was discarded, and the RNA precipitate was air-dried and resuspended in approximately 15-25 µL TE buffer or RNA-free water. The absorbance was measured at 260 nm and 280 nm to confirm that the 260/280 ratio was greater than 1.8 and the concentration was higher than 50 ng/µL. Total RNA quality was assessed using Agilent 2100 bioanalyzer and then 1 μg of total RNA was quantified using the Qubit High-sensitivity kit (Life Technologies, Q32852), and the RNA-seq library was constructed by NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, E7530L).  There were two biological replicates per treatment were sequenced in a HiSeq X-Ten (Illumina, PE150) by Novogene Co. (Beijing)