title: GSM8682269: RMC219-EV.H3K27ac.WI-22008_S40; Homo sapiens; ChIP-Seq
library_construction_protocol: After removing culture media and adding 20 mL of PBS, crosslinking was performed using 16% Paraformaldehyde (Thermo Scientific REF 28908) with a final concentration 1% for 10 minutes at room temperature. Reaction was stopped using 2 mL 10X Glycine for 5 minutes at room temperature. Cells were then scrapped and transferred into a tube. After a 5-minutes centrifugation at 1,500 rpm, pellet was washed with PBS and resuspended in a PBS solution containing 10% of protease & phosphatase inhibitors. After a 5-minutes centrifugation at 1,500 rpm, pellet was resuspended into a buffer containing 50mM Tris-HCl pH 8, 10mM EDTA, and 1% SDS. Nuclear lysate was obtained after incubation for 10 minutes on ice. Sonication was performed using Bioruptor® Plus for 7 to 10 cycles of 30 seconds on and 30 seconds off. Quality of sonication was checked using a solution of 5M NaCl and RNase A (Novagen #70856) for 30 minutes at 37°C. Then, Proteinase K (Novagen #71049) helped remove any protein overnight at 65°C. DNA was purified using QIAquick PCR purification kit. Quantification of DNA was performed using Nanodrop®. DNA shearing was controlled by electrophoresis: samples were considered if the majority of DNA belonged to 100 and 400 bp. Rubicon ThruPLEX Plasma-Seq library preparation system (p/n R400492)