title: GSM4403475: mESCs.ChIP.Input; Mus musculus; ChIP-Seq
library_construction_protocol: Native ChIP-seq was performed as described44. In brief, 1 × 107 cells were harvested and resuspended in 500 μl Buffer 1 (0.32 M sucrose, 15 mM Tris-HCl pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.5 mM DTT, 0.1 mM EGTA, 0.1 mM PMSF, 1:1000 EDTA-free protease inhibitor cocktail [Roche, #4693132001]) supplemented with 0.1% IGEPAL CA-630 (Sigma, # I8896) on ice for 10 min. The lysates was carefully layered on the top of 1 ml Buffer 3 (1.2 M sucrose, 15 mM Tris-HCl pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.5 mM DTT, 0.1 mM EGTA, 0.1 mM PMSF, 1:1000 PIC). After centrifugation (10,000 ×g for 20 min at 4°C), the nuclei were resuspended in 500 μl Buffer A (0.34 M sucrose, 60 mM KCl, 15 mM HEPES pH7.4, 15 mM NaCl, 4 mM MgCl2, 1 mM DTT, 0.1 mM PMSF, 1:1000 PIC) supplemented with 3 mM CaCl2 and 2 U/μl MNase (NEB, # M0247S) and digested for 15 min at 37°C. Then 5 mM EGTA was added to stop the reaction. After centrifuged at 13,500 ×g for 10 min at 4°C, chromatin was resuspended in 10 mM EDTA (pH 8.0) supplemented with 1 mM PMSF and 1:1000 PIC, followed by rotation at 4 °C for 2-4 h. The mixture was adjusted to 500 mM NaCl and rotated for additional 45 min. After centrifugation, the supernatant containing chromatin was diluted to 100 ng/μl with buffer B (20 mM Tris-HCl pH8.0, 5 mM EDTA, 500 mM NaCl, 0.2% Tween 20) and the fragmentation of chromatin was detected by agarose electrophoresis. A total of 2.5 μg fragmented chromatin was kept as the input. For immunoprecipitation, the reaction mixture, including 20 μg fragmented chromatin, 2 μg anti-H3K9me3 antibody (Abcam, #ab8898), and 50 μl protein A/G dynabeads (GE Healthcare) was incubated overnight with gentle rotation at 4°C. The beads were washed three times with Buffer B, and once with Buffer B without Tween 20 at 4°C for 5 min. The DNA was eluted with 300 μl elution buffer (20 mM Tris-HCl pH8.0, 20 mM EDTA, 0.5% SDS), digested with PK enzyme (NEB, #P8107S), and purified using a MinElute kit (Qiagen, #28006). The ChIP DNA library was constructed by a KAPA HTP/LTP Library Preparation Kit according to the manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM4403475