GSM8683294: PM_CLW_RMC_Tissue_Input_for_H3K27me3.WI_22460; Homo sapiens; ChIP-Seq
Frozen tissue kept on dry ice was placed in tissue pulverizer in liquid nitrogen, then crushed. 200 mg of the powdered tissue were added into the 5 mL PBS solution containing 10% of protease & phosphatase inhibitors (Sigma #P8340, Sigma #P5726, & Sigma #P0044). Crosslinking was performed using 37% Formaldehyde with a final concentration 1.5% (Fisher Scientific #CAS 7732-18-5) for 20 minutes at room temperature. Reaction was stopped using 0.5 mL 10X Glycine for 5 minutes at room temperature. 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. Tissue lysate was obtained using Qiagen tissue homogenizer 25 Hz for 20 seconds. Then, after a 5-minutes centrifugation at 1,500 rpm, pellet was resuspended into a buffer containing 5mM PIPES pH 8, 85mM KCl, and 0.5% NP40. Cell lysate was obtained after incubation for 15 minutes on ice. After a 5-minutes centrifugation at 4,500 rpm, pellet was resuspended into a buffer containing 50mM Tris-HCl pH8, 10mM EDTA, and 1% SDS. Nuclear lysate was obtained after incubation for 10 minutes on ice. Sonication was performed using Bioruptor® Plus for 30 to 40 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)