GSM6820885: NRF2_DMSO_H4ac_rep1; Mus musculus; OTHER
CUT&Tag was performed as described previously23, with an additional step of light fixation to better preserve histone acetylation/TF binding. In brief, 1 × 105 cells were lightly fixed with 0.1% paraformaldehyde 5', neutralized by Glycine 125mM, and washed once with 1 ml of wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine (Sigma-Aldrich), 1× Protease inhibitor cocktail (Roche)). Concanavalin A-coated magnetic beads (Bangs Laboratories) were washed twice with binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM MnCl2, 1 mM CaCl2). 10 µl/sample of beads were added to cells in 400ul of wash buffer and incubated at room temperature for 15 min. Beads-bound cells were resuspended in 100 µl of antibody buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.06% Digitonin (Sigma-Aldrich), 2 mM EDTA, 0.1% BSA, 1× Protease inhibitor cocktail and incubated with indicated antibodies (Supplementary Table 3) or normal rabbit IgG (Cell Signaling) at 4 degrees overnight on nutator. After being washed once with Dig-wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.05% Digitonin, 1× Protease inhibitor cocktail), beads-bound cells were incubated with 1 µl Guinea pig anti-rabbit secondary antibody (Antibodies Online ABIN101961) and 2 µl  Hyperactive pA-Tn5 Transposase adapter complex in 100 µl Dig-300 buffer (20 mM HEPES•NaOH, pH 7.5, 0.5 mM Spermidine, 1× Protease inhibitor cocktail, 300 mM NaCl, 0.01% Digitonin) at room temperature for 1 h. Cells were washed three times with Dig-300 buffer to remove unbound antibody and Tn5 and then resuspended in 300 µl of tagmentation buffer (10 mM MgCl2 in Dig-300 buffer) and incubated at 37 °C for 1 h. 10 µl of 0.5 M EDTA, 3 µl of 10% SDS and 5 µl of 10 mg/ml Proteinase K were added to each sample and incubated at 50 °C for 1 h to terminate tagmentation. DNA was purified using chloroform isoamyl alcohol (Sigma Aldrich) and eluted with 25 µl ddH2O. For H3K27ac, 2ul of SNAP-ChIP K-AcylStat panel nucleosomes (EpiCypher) was added as spike-in control at the primary antibody incubation step. For H4ac, 5000 S2 Drosophila cells were added at the cell-bead binding step. For library amplification, 21 µl of DNA was mixed with 2µL i5 unique index primer (10 µM), 2 µL i7 unique index primer (10 µM) and 25 µL NEBNext High-Fidelity 2X PCR Master Mix (NEB) and subjected to the following PCR program: 72℃, 5 min; 98℃, 30 sec; 13 cycles of 98℃, 10 sec and 63℃, 10 sec; 72℃, 1 min and hold at 10℃. To purify the PCR products, 1.1× volumes of pre-warmed Ampure XP beads (Beckman Coulter) were added and incubated at room temperature for 10 min. Libraries were washed twice with 80% ethanol and eluted in 20 µl of 10 mM Tris-HCl, pH 8.  Libraries were sequenced on an NextSeq 550 platform (Illumina, 75 cycles High Output Kit v2.0) and 75-bp paired-end reads were generated. To determine global level differences in histone acetylation signal we used spike-in controls.  Files resulting from 4 sequencing lanes were concatenated into a single file for each condition.