Anti rabbit mab igg isotype control

CUT&RUN assays were performed using the CUT&RUN kit from Cell Signaling Technology (#86652) according to the manufacturer's instructions. Briefly, MDA-MB-231 cells were plated in Cultrex-coated 6-well plates at a density of 300,000 cells/well, and were harvested 5 days later using Cultrex 3D-Culture Cell Harvesting Kit (Trevigen). The resulting single-cell suspensions were collected (10⁵ cells/reaction), washed, and rotated with Concavalin A magnetic beads for 2 hours at 4°C with the following antibodies as indicated: (i) anti-trimethyl-Histone H3 (2 μL/reaction; Cell Signaling Technology #9751); (ii) anti-rabbit mAb IgG Isotype control (5 μL/reaction; Cell Signaling Technology #66362); or (iii) anti-TRIM28 (5 μL/reaction; Thermo Fisher Scientific #MA1-2023, RRID:AB_2209892). Subsequently, the reactions were rotated with pAG-MNase enzyme, which was activated by addition of CaCl₂ and incubated sequentially for 30 minutes at 4°C and 37°C. The resulting DNA fragments were purified with Qiagen QIAquick PCR purification kit (#21804). Input samples underwent DNA extraction and sonication (5 Watts, 30-second intervals for 25 cycles) before purifying DNA and performing qRT-PCR as described previously.