H3k4me1 antibody

The MLL1 complex was prepared by mixing MLL1_3754–3969, WDR5_23–334, ASH2L_FL and RBBP5 at the molar ratio of 1:1:1:1 and incubate on ice for 30 minutes without further purification. The reaction assays were performed with 20 μM SAM, 1 μM nucleosome and 1 μM MLL1 complex in the buffer containing 20 mM HEPES, pH 7.8, 10 mM NaCl and 5 mM DTT. The reaction was incubated at 25°C for 1 h. Each reaction mixture was divided into three parts that were separated on a 15% SDS-PAGE. The methylation products of H3K4 were detected by western blot using corresponding antibodies (H3K4me1 antibody, #39297, Active motif; H3K4me2 antibody, # 07-030 Millipore; H3K4me3 antibody, #9751, CST).

For ULI-NChIP-seq, 10 000 or 40 000 cells were used per reaction. The ULI-NChIP procedure was performed as previously described (17 (link),18 ). Briefly, 2 μg of H3K27ac antibody (39133, Active Motif), H3K4me1 antibody (39297, Active Motif), H3K4me3 antibody (9727, Cell Signaling Technology), H3K27me3 antibody (9733, Cell Signaling Technology) or H3K9me3 antibody (39161, Active Motif) was used for each immunoprecipitation reaction. The sequencing libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645, New England Biolabs) following the manufacturer's instructions. The CUT&Tag assays for CTCF were performed using 50 000 sorted ESCs and 2CLCs as previously described (19 (link)). Two or three replicates were performed. Barcoded libraries were pooled and sequenced on an Illumina HiSeq X Ten platform in the paired-end mode.