GSM5799638: WT; Mus musculus; ChIP-Seq
Cortical neuronal nuclei sorting and H3K4me2 ChIPseq were performed as previously described (Jiang et al., 2017; Kundakovic et al., 2017). Each sample was the cortex pooled from three mice in the same group. In brief, frozen brain tissue was placed in a douncer containing a hypotonic lysis solution and homogenized, followed by ultracentrifugation and resuspension of the nuclei pellet in 1 ml Dulbecco's phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA) and 1:1,000 anti-NeuN antibody (Alexa Fluor 488 conjugated, Millipore MAB377X). The nuclei were incubated for 45 minutes at 4°C, shielded from light. DAPI (1:1,000 dilution) was added to the nuclei prior to sorting. Nuclei were sorted into DPBS to separate NeuN+ and NeuN- populations, followed by pelleting for ChIP experiments. All sorting was done at the Flow Cytometry Center at Mount Sinai. Immediately following nuclei sorting, NeuN+ and NeuN- populations were digested with MNase primarily into mononucleosomes. MNase digestion was stopped with 0.5M EDTA, and the sample was either placed on ice for further processing or frozen at -80C for further use. Next, the sample was treated with a hypotonic solution and mild detergent, and H3K4me2 antibody (Cat#ab7766, Abcam) was added for overnight incubation. H3K4me2 enriched genomic DNA was captured with Protein A/G magnetic beads (88803, Thermo Fisher Scientific), followed by a wash step, elution, and treatment with RNase A and Proteinase K. DNA was purified via phenol-chloroform extraction followed by ethanol precipitation. Following ChIP, library preparation was done to prepare the enriched DNA for sequencing. The steps include: blunting DNA fragments, adding a 3' poly-A tail, ligation of barcoded adaptors, and PCR amplification of ligated DNA. The libraries were then run on an Agilent 2100 Bioanalyzer to check for fragment size. A successful library preparation consisted of a sharp 275bp fragment (150bp for the mononucleosome fragment length + 125bp for adaptor length). The sequencing was performed at the Genomics and Bioinformatics Core of State University of New York at Buffalo.