title: GSM8791672: Mouse Ccne1 4C DMSO; Mus musculus; OTHER
library_construction_protocol: Approximately 3 million C2C12 cells were cross-linked with 2% (v/v) formaldehyde for 10 min at room temperature and quenched by 0.125 M glycine. The samples were lysed in ice-cold lysis buffer [50 mM tris-HCl (pH=7.5), 150 mM NaCl, 5 mM EDTA, 0.5% NP40, 1% Triton X-100 with complete proteinase inhibitors] on ice for 20 min. Samples were then centrifuged at 700g for 5 min. Pelleted nuclei were washed once with 400 μl 1.2x Dpn II buffer [dilute 10x Dpn II buffer by H2O]. The supernatant was discarded, the nuclei were resuspended in 500 μl Dpn II buffer, and 15 μl 10% SDS was added followed by incubation at 37°C for 1 hour with 900 rpm shaking. After incubation. 75 μl of 20% Triton X-100 was added to quench the SDS and then incubated at 37°C for 1 hour with 900 rpm shaking. 200 units of Dpn II restriction enzyme (NEB, R0543) was added, and chromatins were digested at 37°C for overnight. Samples were incubated at 65°C for 20 min to inactivate Dpn II and then cooled to room temperature. 5.7 mL H2O, 700 μl 10x NEB T4 DNA ligase buffer and 3350 units of NEB T4 ligase (NEB, M0202) were added to samples and mixed by swirling at room temperature for overnight. 300 μg of proteinase kinase (Invitrogen, AM2548) was added to samples and incubate at 65°C water bath for overnight. 300 μg of RNase A (Thermo Scientific, EN0531) was then added to samples and the samples were incubated at 37°C for 45 min. The samples were subjected to phenol/chloroform/isoamyl extraction and ethanol precipitation and finally resuspended in 150 μl of 10 mM tris-HCl (pH=7.5). The purified DNA samples was then mixed with 50 μl rCutSmart (NEB, B6004), 295 μl H2O and 50 units of Nla III (NEB, R0125). The mixture was incubated at 37°C with shaking at 500 rpm shaking on the ThermoMixer overnight. The Nla III restriction enzyme was inactivated by incubating at 65°C for 25 min. A ligation mix containing 12.1 mL H2O, 1.4 mL NEB T4 DNA ligase buffer and 100 units of T4 ligase was added to samples, and samples were incubated at room temperature overnight. The samples were purified using phenol/chloroform/isoamyl, precipitated with ethanol, and dissolved in 250 μl tris-HCl (pH=7.5). Finally, the DNA was then purified with QIAquick PCR Purification Kit (QIAGEN, 28104). A total of 800 ng of purified DNA were used as template for 20 cycles of PCR with Phanta Master Mix (Vazyme, P51101), and the PCR products were purified with NucleoSpin Gel and PCR Clean-up kit (MACHEREY-NAGEL, 740609). For DNA library construction, a NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB, E7645S) was used according to the manufacturer's instructions.