GSM7099140: HeLa CENP-A TAP, siNT, Rb CENP-A; Homo sapiens; OTHER
For each CUT&RUN sequencing experiment, 4 x 10^5 siRNA treated HeLaCENP-A-TAP cells were harvested at RT and resuspended in Wash Buffer (20mM HEPES-NaOH pH 7.5, 150mM NaCl, 0.5mM spermidine, 1X Roche protease inhibitor cocktail). HeLaCENP-A-TAP cells were incubated with 10uL Concanavalin A-coated magnetic beads (BioMag) in Binding Buffer (20mM HEPES-KOH pH 7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2 for 7m with rotation. The bead-cell slurry was permeabilized in Antibody buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.0025% digitonin, 2 mM EDTA, 0.1% BSA, 100 nM TSA, 0.1 unit/ml citrate synthase, 1 mM Oxaloacetic acid, 1x Roche protease inhibitor cocktail) and incubated with anti-CENP-A antibody (Antibody 1 = Millipore 07-574, Antibody 2 = Invitrogen MA1-20832) or IgG as a control in Antibody Buffer overnight at 4℃. Following two washes in Digitonin Buffer, beads were incubated in 50uL Digitonin Buffer (20mM HEPES-NaOH pH 7.5, 150mM NaCl, 05mM spermidine, 0.0025% digitonin, 1X protease inhibitor cocktail ) with 1x CUTANA pAG-MNase (EpiCypher 15-1016) for 1h at 4℃. The beads were washed twice in Digitonin Buffer followed by a wash in Low-Salt Wash Buffer (20mM HEPES-NaOH pH 7.5, 0.5mM spermidine, 0.0025% digitonin, 1X protease inhibitor cocktail). The beads were then incubated with ice-cold Calcium Incubation Buffer (3.5 mM HEPES-NaOH pH 7.5, 10 mM CaCl2, 0.0025% digitonin) for 30 min at  0℃. Beads were resuspended with EGTA-Stop Buffer (170 mM NaCl, 20 mM EGTA, 0.0025% digitonin, 50 μg/ml RNase A, 50pg/ml CUTANA E. coli Spike-in DNA – EpiCypher 18-1401) and incubated for 30 min at 37℃. To extract the DNA, 200ul of Oligo Binding Buffer (Zymo) was added to each sample and the total volume was then added to a Zymo-Spin DCC-5 column and centrifuged. The column was washed twice with DNA Wash buffer from the DNA Clean & Concentrator-5 Kit (Zymo). The DNA was eluted from the column in 15ul of DNA Elution Buffer and sample concentration was measured using a Qubit 3.0 fluorometer. CUT&RUN libraries were prepared using an NEBNext Ultra II Library Kit for Illumina and NEBNext Multiplex Oligos for Illumina (Dual Index Primer Set 1) with modifications to manufacturer's protocol. Modifications: End Prep heat inactivation step was conducted at 50°C for 60 min. TheNEBNext Adaptor was diluted 1:25 in 10 mM Tris–HCl, pH 8.0 with 10 mM NaCl. After Adaptor Ligation, a DNA cleanup using 1.75× volumes of Sera-Mag Select beads was conducted. The PCR cycle conditions were programmed as following: 1 cycle: 98°C for 30s, 12cycles: 98°C for 10s, 65°C for 30s, 1 cycle: 65°C for 5m, Hold:4°C. After PCR amplification, DNA cleanup was conducted twice with 1.2× volumes of Sera-Mag Select (Cytiva) beads. Quality control was conducted on the resulting 15uL elution using Agilent 4200 TapeStation D1000 ScreenTape to determine sample concentration and sample quality.