GSM7729718: BM neutrophils, WT, H3Cit, control, rep1; Mus musculus; OTHER
2 × 10^5cells were washed in 200 μL of wash buffer [20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, and 1× protease inhibitors] by gentle pipetting, and then resuspended in 1.5 mL wash buffer. Ten microliters of concanavalin A–coated magnetic beads (Bangs Laboratories, ca.) were activated and then added to 2 × 105cells with incubation on an end-over-end rotator at room temperature for 10 min. The supernatant was removed, and bead-bound cells were resuspended in 50 μL of antibody buffer [20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, one Roche Complete Protease Inhibitor EDTA-Free tablet, 0.05% digitonin, 0.1% BSA, and 2 mM EDTA]. Rabbit polyclonal Anti-Histone H3 (citrulline R2 + R8 + R17) antibody (Abcam, ab5103) was added to bead-bound cells (1:50 dilution) and incubated on a nutator overnight at 4°C. As a control, histone H3 (acetyl K27)  (Abcam ab4729) was added to bead-bound cells (1:50 dilution) and incubated on a nutator overnight at 4°C. After removing the supernatant in the next morning, the cells were incubated with Donkey Anti-Rabbit Immunoglobulin G (IgG) antibody (1:100 dilution, Jackson Immunoresearch) in 100ul antibody buffer on a nutator at room temperature for 1 hour. The control cells were incubated with Rabbit Anti-Mouseimmunoglobulin G (IgG) antibody (1:100 dilution, Jackson Immunoresearch). Bead-bound cells were washed twice with 1mL dig-wash buffer [20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, 1× protease inhibitors, and 0.05% digitonin] to remove the unbound antibodies. 2.5 μL of CUTANA™ pAG-Tn5 (EpiCypher) was added into 50 μl of dig-300 buffer [20 mM HEPES (pH 7.5), 300 mM NaCl, 0.5 mM spermidine, one Roche Complete Protease Inhibitor EDTA-Free tablet, and 0.01% digitonin], and was mixed with the bead-bound cells and then rotated at room temperature for 1 hour. Bead-bound cells were washed twice in 1mL of dig-300 buffer to remove unbound pA-Tn5 protein. Next, cells were resuspended in 300 μl of tagmentation buffer [20 mM HEPES (pH 7.5), 300 mM NaCl, 0.5 mM spermidine, 1 Roche Complete Protease Inhibitor EDTA-Free tablet, 0.01% digitonin, and 10 mM MgCl2] and incubated at 37°C for 1 hour. To stop tagmentation and solubilize DNA fragments, 10 µL 0.5M EDTA, 3 µL 10% SDS and 2.5 µL 20 mg/mL Proteinase K (Thermo Fisher Scientific) were added to each sample, and were further incubated at 55°C for 60 min. To extract the DNA, cells were mixed with 300 µL PCI (Thermo Fisher), centrifuged at 16,000 x g for 3min, and then mixed with 300 µL Chloroform (Sigma). The aqueous layer was added into 300 µL 100% ethanol, centrifuged at 4 °C 16000 x g for 10 min, and then rinsed with 1 ml 100% ethanol. The DNA was dissolved in 30 µL 1 mM Tris-HCl (pH 8), 0.1 mM EDTA. To generate the sequencing libraries, the mixture was mixed with a universal i5 primer and a uniquely barcoded i7 primer and then amplified with the NEBNext HiFi 2x PCR Master mix (NEB, M0541). The libraries were cleaned-up with SPRI paramagnetic beads (Beckman Coulter) and washed with 80% ethanol, and then dissolved in 25 µL 10 mM Tris-HCl (pH 8). TapeStation D1000 capillary electrophoresis was performed to Determine the size distribution and concentration of libraries. Paired-end Illumina sequencing were performed on the barcoded libraries following the manufacturer's instructions.