GSM6564220: H3A31S-GFP Replicate 2; Drosophila melanogaster; MNase-Seq
Resuspended cells were fixed for 5 minutes upon the addition of formaldehyde to a final concentration of 1%. The reaction was quenched by adding 1/10 the volume of 1.25M glycine, followed by end-over-end rotation for 5 minutes. Next, the fixation solution was removed by pelleting cells at 1,320rpm for 7 minutes at 4°C. The cell pellet was washed with 500uL cold 1XPBS, followed by centrifugation and wash removal. This was repeated once. After the last wash, cells were resuspended in 20uL 1XPBS and 1uL of cells was removed for cell count with Trypan Blue.    The cleaved DNA was purified from the cell lysate following the manufacturer's recommendations in the MinElute Reaction Cleanup Kit. The DNA was eluted in 10uL of sterile 10mM Tris, pH8.0.      For a single biological replicate, the ProteinA-MNase and antibody complex was prepared by combining 1uL anti-GFP antibody (Abcam ab290), 3uL PA-MNase enzyme, and 4uL antibody binding buffer (1X Tris EDTA, pH7.5, 150mM NaCl, 0.1% Triton-X 100). The mixture was incubated on ice for 30 minutes. Meanwhile, the cell pellet was resuspended in 500uL RIPA buffer (1X Tris, EDTA pH7.5, 150mM NaCl, 0.2% SDS, 0.1% Sodium Deoxycholate, 1% Triton-X 100)  and incubated at room temperature for 10 minutes. Next, the cells were pelleted by spinning at room temperature for 5 minutes at 3,000 rpm. The cells were washed once with 500uL antibody binding buffer after RIPA removal. The cells werecentrifuged again and the pellet resuspended gently in 100uL antibody binding buffer. The 100uL of cells were gently added to the tubes containing 8uL of the antibody-MNase enzyme complex before incubating on ice for 1hour.    Following incubation, the cells were washed several times to remove excess antibody-enzyme complex. All centrifugation steps were done at 3,000 rpm for 5 minutes at 4°C. First, the cells were spun down to remove the antibody binding buffer. They were then washed in 500uL high salt wash (1X Tris, EDTA pH 7.5, 400mM NaCl, 1% Triton-X 100), with a 1 minute room temperature incubation prior to centrifugation. The high salt wash was repeated a total of three times. After the third wash, the cells were washed in 200uL rinsing buffer (10mM Tris pH7.5, 10mM NaCl, 0.1% Triton-X 100) followed by immediate centrifugation.     After removing the rinsing buffer, the cell pellet was resuspended in 40uL RSB (20mM Tris pH7.5, 10mM NaCl, 2mM CaCl2, 0.1% Triton-X 100). Next, the cells were incubated in a 37°C waterbath for precisely 3 minutes to perform the MNase digestion. To stop the digestion, 80uL of stop buffer (20mM Tris pH8, 10mM EGTA, 20mM NaCl, 0.2% SDS) was added to the reaction. Finally, 1uL of 19mg/mL Proteinase K was added and the solution incubated overnight at 65°C. Library construction was performed by following the manufacturer's recommendations in the NEBNext DNA UltraII kit.