ChIP-seq of Mus musculus: pooled hippocampi from four kainic acid-treated B6 mice analyzed with gamma-H2AX antibody
ChIP-seq of NPAS4 and H2AX was performed using a CUT&RUN assay kit (#86652, Cell Signaling Technology). Fresh hippocampus tissue specimens from 4 or 5 mice (around 50 mg) were pooled and incubated with 4 mL of 0.1% formaldehyde for 2 min at room temperature. The crosslinking reaction was stopped by adding 400 micro-L of 10 glycine and incubating for 5 min with gentle rotation. After centrifugation (2,000 g, 5 min, 4C), supernatants were removed and 1 mL of 1 PBS plus protease inhibitor cocktail (PIC) solution (1:100 volume) added to lyse the tissue. Following further centrifugation (2,000 g, 5 min, 4C), pellets were transferred to Dounce type homogenisers with 1 mL 1 wash buffer containing spermidine and PIC. Homogenised pellets were transferred to 1.5 mL tubes and subjected to centrifugation (3,000 g, 5 min, 4C) to remove supernatants. Pellets were again lysed with 100 micro-L 1 wash buffer and centrifuged (3,000 g, 5 min, 4C). After removal of supernatants, 100 micro-L 1 wash buffer was added. Then, suspension solutions were mixed with 10 micro-L concanavalin A-containing beads in activation buffer and incubated for 5 min at room temperature. Tubes were placed onto a magnetic separation rack and the supernatant removed. After addition of 100 micro-L antibody binding buffer containing spermidine, PIC, and digitonin, lysates were incubated with beads and 10 micro-L anti-NPAS4 antibody or anti-gammaH2AX antibody overnight at 4C with rotation. Next, tubes were placed onto the magnetic stand and the supernatant removed, followed by addition of 1 mL of digitonin buffer, containing spermidine, PIC, and digitonin, and mixing. After removal of the supernatant, 50 micro-L pAG-MNase solution was added and incubated for 1 h at 4C with rotation. After washing the samples with 1 mL digitonin buffer, samples in 150 micro-L digitonin buffer containing spermidine and PIC were placed on ice for 5 min, and incubated with 3 micro-L calcium chloride for 30 min. Samples were then incubated with 150 micro-L 1 Stop buffer for 10 min at 37C. After centrifugation (16,000 g, 2 min, 4C), tubes were again placed onto magnetic stand for 2 min; the supernatants contained ChIPed-DNA. To eliminate the crosslink reaction, 3 micro-L 10% SDS solution and 2 micro-L proteinase K solution (20 mg/mL) were added, followed by incubation at 65C for 2 h. After checking the temperature of solution had reached room temperature, DNA samples were purified using DNA Purification Buffer and Spin Columns (#14209, Cell Signaling Technology).