GSM5743703: H3K4me3 ChIP in WT decidualized stromal cells; Mus musculus; ChIP-Seq
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. The ChIP libraries were prepared using KAPA HyperPrep Kits (Roche, 07962347001) and then run on the Illumina sequencer Hiseq-Xten PE150 using standard Illumina protocols