GSE191327
H3K4me3 ChIP_Seq in mouse decidua
To detect the direct target genes of H3K4me3 in decidual stromal cells, decidual stromal cells are collected and subjected to ChIP-Seq. After aligned to mouse mm10 by STAR, peaks are called by MACS2. We found that H3K4me3 was significantly enriched at the transcription start site (TSS) of expressed genes (RPKM>1). A direct comparison of Menin and H3K4me3 ChIP-seq peaks at gene promoters showed a significantly strong positive correlation (R2=0.5888291). The promoters of 6,800 genes bound by Menin (92% of all genes with promoter bound by Menin) were also H3K4me3 modified. Overall design: Chromatin immunoprecipitation (ChIP) was modified and performed according to the previous described standard protocol. Decidual tissue wetted with PBS quickly cut into small pieces with an iris scissor. The pieces should not be bigger than 0.5 cm3. Snipped tissue was fixed with 1% formaldehyde (CST) for 10 mins at RT (for H3K4me3 ChIP), or fixed with disuccinimidyl glutarate (DSG, Santa Cruz, 2 mM) for 30 mins at RT, washed twice with PBS and then double fixed with 1% formaldehyde for another 10 mins at RT (for Menin ChIP). Fixation was stopped by adding glycine (0.125 M). Tissue homogenate obtained by using Dounce Tissue Grinder was filtered through a 200 µm cell strainer to remove connective tissue. Fixed cells were lysed by Lysis Buffer 1 (50 mM HEPES pH 7.5, 1 mM EDTA, 140 mM NaCl, 0.5% NP-40, 10% glycerol, 0.25% Triton X-100) and Lysis Buffer 2 (10 mM Tris-HCl pH8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl) and Lysis Buffer 3 (10 mM Tris-HCl pH8.0, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 0.1% Sodium Deoxycholate, 0.1% N-lauroylsarcosine). All lysis buffers should be supplemented with protease inhibitors cocktail (Roche) before use. Chromatin DNA was sheared to 300-500 bp average in size by using a BioRuptor sonicator (Diagenode). Solubilized chromatin was was centrifuged to remove debris and incubated overnight at 4°C with antibody against Menin (Bethyl, 10 µg) and H3K4me3 (Abcam, 1 µg) bound to 20 µl protein A magnetic beads (Invitrogen). After washing and elution, the protein-DNA complex was reversed by heating at 65°C overnight. Immunoprecipitated DNA was purified by using QIAquick spin columns (Qiagen). Immunoprecipitated and input DNA were quantified using Qubit 4.0 fluorometer. ChIP-seq libraries were prepared by using the KAPA DNA Hyper Prep Kit (KK8502) following the manufacturer's instruction and sequenced with an Illumina Nova PE150. For ChIP-qPCR, chromatin was sheared by sonication until the average length of 500-1000 bp.