title: GSM8663320: AG14980, Wildtype, 0.4%DMSO for 24h, RNAseq rep3; Homo sapiens; RNA-Seq
library_construction_protocol: The collected cells were lysed in 1 ml of Trizol at room temperature (RT) for 5 minutes. Subsequently, 200 μl of chloroform was added, and the samples were centrifuged at 12,000 g for 15 minutes at 4°C to separate the phases. Prior to centrifugation, the samples were vortexed for 15 seconds and incubated at RT for 3 minutes. The upper phase, containing RNA, was carefully transferred to a new tube and mixed with 500 μl of isopropanol. The RNA was then precipitated at -20°C for 2 hours and subsequently pelleted at 12,000 g for 30 minutes at 4°C. The supernatant was discarded, and the RNA pellet was washed with 1 ml of ice-cold 75% ethanol. After centrifugation at 9,000 g for 5 minutes at 4°C, the ethanol wash step was repeated, and residual ethanol was aspirated using a pipette. The RNA pellet was air-dried by leaving the tubes open on the counter for approximately 5 minutes.The air-dried RNA pellet was resuspended in 50 μl of DNase reaction mix (5 μl of 10X Reaction Buffer for Turbo DNase, 1 μl of TURBO™ DNase (Thermo Fisher Scientific, AM2238) , 0.5 μl of recombinant RNase inhibitors (Jena Bioscience, PCR-392S) , and 43.5 μl of RNase-free water). The samples were incubated at 37°C for 25 minutes with gentle agitation at 550 rpm to carry out DNAse treatment. For reextraction of RNA, 150 μl of H2O and 200 μl of Phenol/Chloroform/Isoamylalcohol were added. The samples were vortexed for 15 seconds and then centrifuged at 13,000 rpm for 2 minutes at 4°C. The upper phase was transferred to a new tube and mixed with 20 μl of 3M Na-acetate pH 5.5 and 200 μl of isopropanol. The samples were incubated at -20°C for 1 hour to allow for the second RNA precipitation. The RNA was then pelleted, and the washing procedure was repeated as described in the initial precipitation step. The RNA pellet obtained after the final precipitation steps was dissolved in H2O. poly A mRNA enrichment kit (New England Biolabs, E7490) and RNAseq library preparation kit (New England Biolabs, E7760S), following the manufacturer's protocols
identifiers.namespace: GEO
identifiers.id: GSM8663320_r1