CENP-A CUT&RUN (low-salt fraction) of Homo sapiens: CHM13 Expt 1
CUT&RUN was carried out as in Thakur and Henikoff 2018, with some variations. Frozen pellets of 1,6M HG002 cells or 1,2M CHM cells were thawed on ice and centrifuged at 500g for 5 minutes at 4C. Cells were washed with cold PBS twice. For nuclear extraction cell pellet was resuspended in 500 mL of Nuclear Extraction Buffer (NEB, 20mM HEPES pH 7.9, 10mM KCl, 0.5mM Spermidine, 0.1% NP40, 20% glycerol, Roche Proteinase Inhibitor tablets) by repipetting gently, and incubated on ice for 5 minutes. Cells were centrifuged and washed with Washing Buffer (WB, 20mM HEPES pH 44 7.5, 150mM NaCl, 0.5mM Spermidine, 0.1% BSA, 0.05% NP40, Roche Proteinase Inhibitor tablets), blocked in WB containing BSA, and incubated in primary antibody for 2h at 4C under rotation. Cells were washed twice with WB and incubated with pAG-MNase (Cell Signaling) for 1h at 4C under rotation. For pAG-MNase digestion, samples were incubated on ice-water (0C) for 10 minutes, and CaCl2 was added to 2mM as final concentration. Samples were incubated for 30 minutes in cold (0C). To stop digestion, an equal volume of 2X STOP solution (200mM NaCl, 20mM EDTA, 4mM EGTA, 0.1% NP40) was added. To recover low-salt fragments, samples were incubated for 1h at 4C under rotation, centrifuged at 500g for 5 minutes and supernatant collected and labeled as low-salt fraction. To recover high-salt fragments, the pellet was resuspended in 150 mL of low-salt solution (175mM NaCl, 20mM EDTA, 4mM EGTA, 0.1% NP40) and then 150 mL of high-salt solution (825mM NaCl, 20mM EDTA, 4mM EGTA, 0.1% NP40) was added. Cells were rotated for 1h at 4C, centrifuged at 16000g for 5 minutes and supernatant collected and labeled as high-salt fraction. RNase A was added to both fractions following incubation for 20 minutes at 37C . Samples were treated with Proteinase K for 1h at 65C (or overnight), and DNA extraction was carried out with MasterPure Complete DNA Isolation kit (Lucigen) as indicated by the manufacturer. Samples were analyzed by a Fragment Analyzer. For library preparation, 1.5 pg of Spike-in Yeast DNA was added (obtained from the Henikoff lab) and NEBNext Ultra II End repair/A-tailing and Ligation kits were used as indicated by the manufacturer. DNA was cleaned using AMPure XP beads and the PCR reaction was carried out using NEBNext UltraII Q5 master mix and NEBNext multiplex oligos for Illumina (12 cycles with annealing/extension for 15 seconds at 65C). Libraries were cleaned using AMPure XP beads as indicated by the manufacturer. Libraries were sequence using NovaSeq 50PE sequencing. Primary antibodies used were: mouse a CENP-A (Abcam, ab13939), rabbit a CENP-B (Abcamab25734).