Cl 18

Mice were sacrificed by cervical dislocation. Brains were removed and the somatosensory cortex (+0.38 mm to −1.94 mm anteroposterior from Bregma) was isolated. Tissues were snap-frozen and stored at −80°C. Defrosted samples were sonicated for 10 sec (QSonica CL-18; 1 sec alternating on/off pulses, 40% pulse amplitude) in ice-cold buffer containing: 320 mM Sucrose, 10 mM EDTA, 50 mM Tris pH 7.4, protease inhibitor (cOmplete mini, Roche), Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich), Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich). Samples were centrifuged (Sigma 4–16KS, 12 130 rotor) at 1500×g for 20 min at 4°C. The supernatant was transferred to a new tube and centrifuged for 30 min at 16 000×g at 4°C. The supernatant was removed, and the pellet was resuspended in 50 mM Tris containing phosphatase and protease inhibitors (TPP). Protein concentration was measured using a BCA protein assay and samples were normalized to 1 mg/ml in TPP.

Purified ACMs were resuspended in NP40 lysis buffer (0.5% NP40, 25 mM KCl, 3 mM MgCl₂, 10 mM Tris–HCl, pH 8.0) and homogenized until nuclei were released from the cytoskeleton. Extraction buffer was supplemented with 1 mM Na₃VO, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride and 1× Protease Inhibitor Cocktail (Millipore 539134). The homogenate was centrifuged with 50×g for 1 min at 4 °C and supernatant (enriched nuclear) was corrected. The enriched nuclear was pelleted with 800×g for 10 min at 4 °C. Nuclear pellets were resuspended in SDS/βME nuclear buffer (1% SDS, 25 mM 2-mercaptoethanol, 137 mM NaCl, 0.5% NP40, 25 mM KCl, 3 mM MgCl2, 10 mM Tris–HCl, pH 8.0) and sonicated with probe sonicator (Qsonica, CL-18) with 25% power for 10 s 2 time (30 s interval). Nuclear extracts were cleared by centrifugation at 20,000×g for 10 min at 15 °C. DNA concentration was measured using Quant-iT™ PicoGreen^® dsDNA Reagent (Life technology) and used to normalize samples. Nuclear extract (50–250 ng DNA) were separated by SDS–PAGE, transferred to polyvinylidene fluoride membrane and probed using specific primary antibodies and appropriate HRP-conjugated secondary antibody for ECL detection. Antibodies used are listed in Additional file 3.

A Fisherbrand^™ Sonic Dismembrator (Model 120) equipped with a single-tip Qsonica CL-18 or a 602-A 8-tip sonicator probe was used to perform sonication. The ELISA samples were analyzed with a BioTek Synergy LX plate reader set to 450 nm. The PicoProbe^™ assay samples were analyzed with a BioTek Synergy LX plate reader installed with a red filter cube (Excitation/Emission: 530/590 nm; P/N: 1505004).