GSM7767052: E14_mESC_reChIP_K4K27_R1; Mus musculus; ChIP-Seq
Cells were fixed with 1% formaldehyde in prewarmed DMEM for 8 minutes at room temperature. After quenching with 1M glycine, cells were washed by ice cold 1X PBS and resuspend in chromatin resuspension buffer (PBS + 0.5M EDTA). Chromatin pellets were snap-freeze on dry ice and store at -80 degree freezer before use. ChIP-re-ChIP was performed on 2 million cell pellets. Briefly, cell pellets were gently lysed and treated with MNAse to generate predominantly mononucleosomes then chromatin pre-cleared to reduce non-specific binding by incubating with washed dynabeads for 3 hours at 4 degrees celcius. During this time, three sets of antibody-dynabeads are formed for the IgG control, H3K4me3 and H3K27me3 immunoprecipitations. 5% of the precleared chromatin was set aside as an input control and the remainder split across the three antibody-dynabead reactions for the first overnight immunoprecipitation.  Following the first immunoprecipitation, the chromatin-antibody-dynabead complexes are thoroughly washed to remove any non-specific binding, prior to chromatin elution with SDS at 37 degrees. SDS was removed and chromatin concentrated with a buffer exchange using  Amicon 3KDa molecular weight filters, setting aside 10% of this sample that represents in-line total H3K4me3 or total H3K27me3 controls. The second immunoprecipitation was then performed overnight using the opposing antibody such that the reChIP was performed in both orientations (H3K4me3 followed by H3K27me3, K4K27 or vice-versa K27K4) with IgG followed by IgG (IgG-IgG) as a negative control.  Chromatin is then eluted, treated with RNAse A and proteinase K, crosslinks reversed and enriched DNA fragments purified. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit.