GSM4705944
GSM4705944: FGF_input_rep1; Mus musculus; ChIP-Seq
Frozen cells were thawed on ice, resuspended in PBS, and cross-linked with 1% formaldehyde for 5 mins at room temperature.  Cross-linking was quenched with 125 mM glycine for 5 mins at room temperature.  Cells were lysed as previously described (Lee et al, 2006), except that N-lauroylsarcosine was used at 0.1%,  and sonicated with a S220 Focused-ultrasonicator (Covaris) for 15 min. Sonicated lysates were supplemented with 1% Triton X-100 and then cleared by centrifugation at full speed in microcentrifuge for 10 min at 4°C. The supernatant was transferred to a new tube and the protein content was measured by Bradford assay. Extracts were incubated overnight at 4°C with 5 ug of anti-H3K27ac (Active Motif) pre-bound to protein G dynabeads (Thermo Scientific) that were pre-blocked with 0.5% BSA in PBS. Beads were washed and then incubated with elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS) for 30 min at 65°C. Crosslink reversal was performed by overnight incubation at 65°C. Samples were diluted with 1 volume of TE and then treated with RNaseA (0.2 mg/ml for 2 hrs at 37°C) and proteinase K (0.2 mg/ml for 2 hrs at 55°C) prior to DNA extraction with phenol:chloroform:isoamyl alcohol. Library preparation was performed using the NEBNextUltra II DNA Library Prep Kit for Illumina (New England Biolabs).
GEO
GSM4705944
GEO Accession
GSM4705944