GSM9157146: kd-siRNA-MIR222HG_3; Homo sapiens; RNA-Seq
To isolate nuclear extract, medium was aspirated and cells were washed once with cold Hank's solution. Harvesting was performed by scraping cells twice in 500 µL Hank's solution and centrifuging (1 min, 13 000 rpm at 4 ◦C). The supernatant was removed and the pellet resuspended in 600 µL buffer A. After incubation on ice for 15 minutes, 45 µL 10 % Nonidet (Sigma Aldrich) were added to lyse the cytosolic membrane, the cells were vortexed for 10 seconds and centrifuged (1 min, 13 000 rpm at 4 ◦C). The supernatant, containing the cytosolic fraction, was removed and the pellet resuspended in 150 µL buffer C. To lyse the nuclei, the resuspended pellet was incubated in a shaker for 15 minutes at 1400 rpm and 4 ◦C. To remove insoluble debris, the lysate was centrifuged (5 min, 13 000 rpm at 4 ◦C) and the supernatant was transferred into a new 1.5 mL tube 900 ng of total RNA isolated from HUVEC was used as input for SMARTer Stranded Total RNA Sample Prep Kit—HI Mammalian (Takara Bio). Sequencing was performed on the NextSeq500 instrument (Illumina) using v2 chemistry, resulting in an average of 34 million reads per library with 1,× 75bp single end setup.