GSM9827166: neurons,WT,week18,male,rep1 (9278_H3K27ac); Mus musculus; OTHER
Cut&Tag was used to profile H3K4me3, H3K4me2, H3K4me1 and H3K27ac in eighteen-week-old cortical neurons. The CUT&Tag-direct V.3 protocol31,32 were performed with minor modifications. For each sample, 50,000 GFP⁺/NeuN⁺ excitatory neuron nuclei were bound to Concanavalin-A beads (3.5 µL bead slurry per reaction) and incubated with primary antibodies (1:50 dilution) overnight at 4°C: H3K4me1 (EpiCypher #13-0057), H3K4me2 (EpiCypher #13-0027), H3K4me3 (Active Motif #39159), or H3K27ac (Active Motif #39133). After washing, bead-bound nuclei were incubated with guinea pig anti-rabbit secondary antibody for 30 min at room temperature. Protein A–Tn5 transposome (pAG-Tn5; EpiCypher #15-1117) was added (2.5 µL per reaction) and incubated for 1 hr at room temperature, followed by tagmentation at 37°C for 1 hour.Tagmented chromatin was released by incubation with 0.1% SDS at 58°C for 1 hr and neutralized with 0.67% Triton-X100. Libraries were amplified using NEBNext High-Fidelity 2× PCR Master Mix (non-hot-start) (NEBNext #M0541L) with unique dual indexes (Buenrostro-style i7/i5 indexing) for 11–12 cycles depending on mark abundance. PCR products were cleaned with 1.3× SPRI bead purification, eluted in 13.2 µL, and assessed by Qubit and Bioanalyzer.