Abe24

ChIP, ChIP-Seq and sequential-ChIP assays were performed as described previously (6 (link),14 (link)). Briefly, Nuclei were sonicated with the Bioruprtor™ UCD200. Chromatin samples prepared from 1 × 10⁶ cells of Day 19 EBs or bone marrow (BM) cells were immunoprecipitated with antibodies specific for histone modifications or epigenetic regulators. The immunoprecipitated chromatin complexes were reverse cross-linked and purified. The recovered DNA was analyzed by qPCR. The relative enrichment was determined by the following equation: 2^{Ct(IP)-Ct(ref)}. The sequential ChIP assays were carried out essentially as described (14 (link),27 (link)) with some modifications. Briefly, chromatin prepared from 5 × 10⁶ cells was immunoprecipitated with antibodies specific for H3K4me3 or USF1. The H3K4me3 or USF1-selected chromatin complexes were eluted, dialyzed and subsequently immunoprecipitated with antibody specific to BPTF. The bound protein–DNA complexes were reverse cross-linked and purified. The recovered DNA was analyzed by qPCR.
Antibodies against H3K4me2 (07-730), H3K4me3 (04-745), SNF2L (05-698) and BPTF (ABE24) were purchased from Millipore, H3K4me1 (ab8895) was from Abcam, ASH2L (A300-489A) and Setd1a (A300-289A) were from Bethyl laboratories and USF1 (H-86) was from Santa Cruz Biotechnology.