GSM8658464: input_wt_NLRP3_rep2; Mus musculus; ChIP-Seq
Brains were  dissociated using the Neural Tissue Dissociation kit (P) (Miltenyi Biotec), followed by removal of myelin and then MACS isolation of CD11b+ microglia following the manufacturer's guidelines (Miltenyi Biotec). For H3K27ac ChIP-seq, fixed (1% formaldehyde for 10 min) nuclei were quickly thawed on ice and resuspended in ice-cold LB3 buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and 1x protease inhibitor cocktail. Chromatin was sheared by sonication using a PIXUL Multi-Sample Sonicator (Active Motif) set at default parameters for a processing time of 48 min. The lysates were adjusted to 250 μl with LB3 and further diluted with 25 μl 10% Triton X-100 (final concentration 1%). Samples were spun down at maximum speed at 4℃ for 10 minutes. For DNA input control, 3 μl of the lysate was taken and volume adjusted to 25 μl with TT (10 mM Tris-HCl pH 8, 0.05% Tween-20) and stored at 4℃ until library preparation. For immunoprecipitation, 20 μl of Dynabeads Protein A and Protein G (ThermoFisher Scientific, 10001D and 10003D, respectively) and H3K27ac antibody (2 μl serum; ActiveMotif, 39135) was added to the diluted lysates and rotated overnight at 4°C. Beads were collected on a magnet and washed three times each with wash buffer I (20 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA), wash buffer III (10 mM Tris/HCl pH 7.4, 250 mM LiCl, 1% Triton X-100, 0.7% Na-Deoxycholate, 1 mM EDTA), twice with ice-cold TET (10 mM Tris/HCl pH7.5, 1 mM EDTA, 0.2% Tween-20), twice with IDTET (10mM Tris-HCl pH 8.0, 0.1mM EDTA, 0.2% Tween-20) and finally resuspended in 25 μl TT. For H3K27ac ChIP-seq, fixed (1% formaldehyde for 10 min) nuclei were quickly thawed on ice and resuspended in ice-cold LB3 buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and 1x protease inhibitor cocktail. Chromatin was sheared by sonication using a PIXUL Multi-Sample Sonicator (Active Motif) set at default parameters for a processing time of 48 min. The lysates were adjusted to 250 μl with LB3 and further diluted with 25 μl 10% Triton X-100 (final concentration 1%). Samples were spun down at maximum speed at 4℃ for 10 minutes. For DNA input control, 3 μl of the lysate was taken and volume adjusted to 25 μl with TT (10 mM Tris-HCl pH 8, 0.05% Tween-20) and stored at 4℃ until library preparation. For immunoprecipitation, 20 μl of Dynabeads Protein A and Protein G (ThermoFisher Scientific, 10001D and 10003D, respectively) and H3K27ac antibody (2 μl serum; ActiveMotif, 39135) was added to the diluted lysates and rotated overnight at 4°C. Beads were collected on a magnet and washed three times each with wash buffer I (20 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA), wash buffer III (10 mM Tris/HCl pH 7.4, 250 mM LiCl, 1% Triton X-100, 0.7% Na-Deoxycholate, 1 mM EDTA), twice with ice-cold TET (10 mM Tris/HCl pH7.5, 1 mM EDTA, 0.2% Tween-20), twice with IDTET (10mM Tris-HCl pH 8.0, 0.1mM EDTA, 0.2% Tween-20) and finally resuspended in 25 μl TT.