Cut run assay kit

To perform ChIP experiments, we utilized PANC-1 cells transfected with L3MBTL2 or control vector. 2×10⁵ cells were harvested for each antibody reaction and additional same amount cells for input samples and negative control Rabbit (DA1E) mAb IgG. The CUT&RUN and ChIP assays was performed according to the instruction (CUT&RUN Assay Kit, Cell Signaling Technology, USA). Lyse the cells and fragment the chromatin by sonicating the input samples using M220 focused-ultrasonicator (Covaris, USA) device. The microTUBE-50 AFA Fiber Screw-Cap (Covaris, USA) was used for sample holder and 150 bp target base pair peak program was chosen for chromatin fragmentation. The peak incident power was 75 W and cycles per burst was set to 200 cpb with the total treatment time was 520s. DNA were purified from input and enriched chromatin samples using the Chromatin IP DNA Purification Kit (Active Motif, USA). Quantitative PCR reactions were performed using the ChamQ SYBR Color qPCR Master Mix (Vazyme, Nanjing, China) on the ABI STEPONE system (Applied Biosystems, Foster City, CA, USA). The antibodies used in this procedure are listed in key resources table and the primers used are listed in Table S1. All ChIP assays were repeated three times and individual qPCR reactions performed in triplicates with results presented as mean ± SD.

For the analysis of histone modification–promotor interactions, cleavage under targets and release using nuclease (CUT&RUN) assays were performed using the CUT&RUN assay Kit (#86652; Cell Signaling Technology) according to the manufacturer's protocol. CCS cells were seeded at 1 × 10⁶ cells per 6-cm dish and cultured for 24 hours, after which they were treated with 3 μmol/L vorinostat or vehicle. For each reaction, 1 × 10⁵ cells were used, and the cells were bound to concanavalin A beads and permeabilized with a digitonin-containing buffer. Antibodies were then applied (shown in Supplementary Table S1) at a dilution of 1:100 and incubated at 4°C overnight. Antibody-bound DNA was purified using DNA purification buffers and spin columns (14209S; Cell Signaling Technology) and amplified for use as templates in CUT&RUN–qRT-PCR or to construct libraries in CUT&RUN-sequencing (CUT&RUN-seq). One-hundred base pair paired-end sequencing was then performed using the NovaSeq 6000 System (Illumina). CUT&RUN-seq data were analyzed by mapping the reads using Bowtie2. The sequencing reads were aligned to human genome build hg38. The UCSC genome browser (12 (link)) was used to visualize the mapped reads.

CUT&RUN assays were performed in MCF7 cells using the CUT&RUN Assay Kit (Cat#86652; Cell Signaling Technology Danvers, MA, USA), according to the manufacturer's instructions. Briefly, 2 × 10⁵ cells were harvested, washed, bound to activated concanavalin A–coated magnetic beads, and permeabilized. The bead–cell complexes were incubated overnight with the appropriate antibody at 4°C. The complexes were washed three times, and the cells were resuspended in 100 μl protein A and G/micrococcal nuclease (pAG/MNase) and incubated for 1 h at room temperature. The samples were then washed three times with digitonin buffer containing protease inhibitors, resuspended in 150 μl digitonin buffer, and incubated for 5 min on ice. MNase was activated by adding calcium chloride, and the samples were incubated at 4°C for 30 min. The reaction was stopped by adding 150 μl stop buffer, and the samples were incubated at 37°C for 10 min to release the DNA fragments. DNA was extracted using the DNA purification columns included in the CUT&RUN Assay Kit. qPCR was performed using UBE2T promoter–specific primers, and relative fold change was calculated as the ratio of immunoprecipitated DNA to IgG-precipitated DNA. The primer sequences and antibodies used for the CUT&RUN assays are listed in Supplementary Table S5.