title: H3K27ac_Sham_WT2
design: CUT&Tag for H3K27ac was performed starting from sciatic dorsal root ganglia NeuN-positive sorted neuronal nuclei 3 days after sciatic nerve crush (SNC) or sham. Sorted nuclei were pelleted by centrifugation, resuspended in wash buffer (20mM HEPES pH 7.5, 150 mM NaCl, 0.5mM spermidine, and EDTA-free protease inhibitors), and bound to 3.5 microliters Concanavalin A beads (Statech Scientific) in binding buffer 20mM HEPES-KOH pH 7.5, 10mM KCl, 1mM CaCl2 and 1mM MnCl2. Nuclei-bound beads were incubated with 1:100 anti H3K27ac (ab4729, Abcam) antibodies for 2 hours at r.t. and then o.n. at 4C in antibody buffer (20mM HEPES pH 7.5, 150 mM NaCl, 0.5mM spermidine, 0.1% BSA, and EDTA-free protease inhibitors). After incubation with 1:100 guinea pig anti-rabbit secondary antibodies (ABIN101961, Antibodies online) for 1 h and washing, nuclei were incubated with 1:20 CUTANA pAG-Tn5 pre-loaded adaptor complex (Stratech) in 300-wash buffer (20mM HEPES pH 7.5, 300 mM NaCl, 0.5mM spermidine, and EDTA-free protease inhibitors) for 2 hours at r.t.. Tagmentation was initiated by adding 10mM MgCl2 and carried out at 37C for 1 h. Tagmented DNA was released by resuspending the beads in 0.1% SDS in 1M TAPS pH8.5. Libraries were generated with 11-12 cycles-PCR using NEBnext PCR mix (New England Biolabs).