GSM4261616: ChIPseq_Zic3_2iL_rep2; Mus musculus; ChIP-Seq
ESCs were fixed using 1% formaldehyde (Millipore #344198) for 10 min at room temperature then quenched by adding 1.25M glycine to a final concentration of 0.125M. Cell pellets were snap frozen and stored in -80℃. Every 10 million cells were lysed by 300μL 1% SDS include freshly made Protease Inhibitor (PI) cocktail (Roche #4693132001), sonicated with Diagenode Bioruptor Pico and diluted with 2.7mL PBA (1x PBS + 0.5% BSA) with fresh added PI cocktail. Dynabeads protein A+G (Invitrogen #10008D, 10009D) were washed and pre-blocked with cold PBA for 30 minutes. Diluted chromatin containing 30μg DNA was incubated with 15μg P53 antibody (Novocastra #CM5P) and 60μL pre-blocked beads and rotated at 4℃ overnight. After incubation, beads were washed subsequently with High-NaCl, Low-NaCl, LiCl, TE and TE washing buffers for 10 minutes and transferred to new eppendorf tube. Beads were eluted with 200µL elution buffer (1%SDS, 0.2M NaCl, 0.1μg/μL Proteinase K) at 65°C thermo shaker 1000rpm for 20 minutes. Supernatants were purified with MinElute PCR Purification Kit (QIAGEN #28006). 1-5ng of DNA was used for library construction with KAPA Hyper Prep Kit (KAPA #KK8504).
GEO Accession
GSM4261616