title: GSM9246995: AML-387919_H3K4me_Plate_2; Homo sapiens; OTHER
library_construction_protocol: Lightly cross-linked nuclei were prepared from cells as described in steps 2–14 of the Bench Top CUT&Tag protocol on protocols.io (https://doi.org/10.17504/protocols.io.bcuhiwt6). In brief, cells were pelleted for 3 minutes at 600g, resuspended in hypotonic NE1 buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.5 mM spermidine, 10% Triton X-100 and 20% glycerol) and incubated on ice for 10 minutes. The mixture was pelleted for 4 minutes at 1,300g, resuspended in 1× PBS and fixed with 0.1% formaldehyde for 2 minutes before quenching with 60 mM glycine. Nuclei were counted using the ViCell Automated Cell Counter (Beckman Coulter) and frozen at −80 °C in 10% DMSO at a concentration of 1M nuclei/mL for future use. Blood and bone marrow samples were collected at presentation from adult patients with de novo AML on an Institutional Review Board–approved banking protocol (#201011766). Nuclei were extracted from samples according to the nuclei preparation protocol listed above with the following modifications: NE1 hypotonic lysis buffer incubation time was optimized for each individual patient sample based on percentage of trypan blue-positive nuclei yield from input cells, with all samples incubated for between 45 seconds-2 minutes. All patient samples were preserved as native nuclei without formaldehyde crosslinking at a maximum concentration of 1M nuclei/mL. Specific samples for which fewer than 1M nuclei were recovered were preserved at a concentration lower than 1M nuclei/mL. CUT&Tag-Direct: CUT&Tag-Direct was carried out as previously described (https://doi.org/10.17504/protocols.io.bcuhiwt6). In brief, nuclei were thawed and bound to washed paramagnetic concanavalin A (ConA) beads (Bangs Laboratories) at a concentration of 5 µL washed beads per 50K nuclei, and then incubated with primary antibody at 4 °C overnight in Wash Buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and Roche Complete Protease Inhibitor Cocktail) with 2 mM EDTA. Bound nuclei were washed and incubated with 1:100 secondary antibody for 1 hour at room temperature and then washed 2x and incubated in Wash-300 Buffer (Wash Buffer with 300 mM NaCl) with 1:50 5 µM loaded pA–Tn5 for 1 hour at room temperature. Nuclei were washed 2x and tagmented in Wash-300 Buffer with 10 mM MgCl2 for 1 hour at 37 °C and then resuspended sequentially in 50 µl of 10 mM TAPS and 5 µl of 10 mM TAPS with 0.1% SDS and incubated for 1 hour at 58 °C. The resulting suspension was mixed well with 16 µl of 0.9375% Triton X-100, and then 2 µL each of F and R primers and 25 µL 2× NEBNext Master Mix (New England Biolabs) were added for direct amplification with the following conditions: (1) 58 °C for 5 minutes, (2) 72 °C for 5 minutes, (3) 98 °C for 30 seconds, (4) 98 °C 15 seconds, (5) 60 °C for 15 seconds, (6) repeat steps 4–5 13 times, (7) 72 °C for 1 minute and (8) hold at 8 °C. DNA from amplified product was purified using 1.1× ratio of HighPrep PCR Cleanup System (MagBio) and resuspended in 25 µl of 10 mM Tris-HCl with 1 mM EDTA, and concentration was quantified using the Qubit (Thermo Fisher Scientific) and TapeStation (Agilent) systems. Plate-CUT&Tag: CUT&Tag-Direct experiments were carried out in individual wells of twin.tec lo-bind 96-well PCR microplates (Eppendorf) using multi-channel pipettes and reservoirs, with the following modifications: After tagmentation and washing with 10 mM TAPS, beads were resuspended individually well-by-well in 16 µL 10 mM TAPS with 0.1% SDS using a single-channel pipette with vigorous pipette mixing. The plate was sealed and vortexed briefly for 10 seconds at 1000 RPM on a Thermomixer (Eppendorf). The sealed plate was spun briefly in a swinging bucket rotor, then denatured on a 96 well deep block thermal cycler (BioRad) for 1 hour at 58˚C. After denaturation, samples were neutralized with 5 µL 10% Triton X-100, and the plate was re-sealed and vortexed briefly for 10 seconds at 1000 RPM on the Thermomixer. Primers and NEBNext 2x Master Mix were then added, mixed, and the plate amplified according to the CUT&Tag-Direct thermal cycler program on a 96 well block. Libraries were purified and eluted as described above.