Anti h3k4me3

The following antibodies targeting histone modifications were used for ChIP-seq (1 μg per ChIP for cell numbers below 100,000, 2 μg per ChIP for cell total cell numbers per ChIP below 250,000): anti-H3K27ac (C15410196, lot A1723–041D), anti-H3K4me3 (C15410003, lot A5051–001P), anti-H3K4me1 (C15410194, lot A1863–001D), anti-H3K36me3 (C15410192, lot A1847–001P), anti-H3K9me3 (C15410193, A1671–001P), anti-H3K27me3 (C15410195, lot A1811–001P), all from Diagenode. Transcription factors antibodies: CTCF (3 μg/ChIP, Abcam, ab70303), p300 (10 μg/ChIP, Santa Cruz, sc-585).

Islets were pooled from 4 adult mice per ChIP experiment performed using minor modifications of the micro-ChIP protocol [31] (link). Islets were lysed with 115 µL of lysis buffer (50 mM Tris-HCl pH 8,0; 10 mM EDTA; 1% SDS; 1 mM PMSF; 20 mM butyrate; protease inhibitors cocktail from Roche) during 20 minutes at 4°C. Samples were then sonicated 3x[20 sec ON/40 sec OFF] and washed with RIPA ChIP buffer (10 mM Tris-HCl pH 7,5; 1 mM EDTA; 1% TX-100; 0,1% SDS; 0,1% Na-deoycholate; 100 mM NaCl; 1 mM PMSF; 20 mM butyrate; protease inhibitors cocktail). 1 µg of anti-H3K4me3 (Diagenode, Denville, NJ, USA) and 10 µL of agarose beads blocked with sonicated salmon sperm (Millipore, Temecula, CA, USA cat #16–157) were used per ChIP sample. After elution of DNA/Protein/antibodies complexes, reversal of the crosslinking and proteinase K/RNAse A treatment, DNA was purified using the NucleoSpin kit from Macherey-Nagel (Duren, Germany). For primers used see Table S1.

Chromatin immunoprecipitation was performed to analyze the enrichment of select regions and confirm H3K4me3 binding at regions of interest using a True MicroChIP Kit (Diagenode) according to the manufacturer’s protocol. A total of 1×10⁶ cells were collected and crosslinked with 1% formaldehyde. Cells were disrupted by ultrasonication to fragment the DNA into 200–500 bp pieces. Specific antibodies to the protein of interest [anti-KDM5c (Abcam) and anti-H3K4me3 (Diagenode)] were added to bind target protein-DNA complexes, and incubated overnight. Protein A agarose was added to bind the antibody-target protein-DNA complexes, washed to remove non-specific binding, and then the enriched target protein-DNA complexes were eluted from the beads and the crosslinks were reversed. After purification, enriched DNA-fragments were subjected to qPCR analysis using fluorescence quantitative PCR. Goat IgG was used as the negative control. The fold change in the amount of the DNA fragment enriched by a specific antibody versus the total input was calculated by the following formula: % recovery = 100^∗2^∧[(Ct(input)-log%(x%)/log2)-Ct(sample)]. The primers used in the ChIP-PCR assays are listed in the Table 1.