GSM8946588: HUVEC (Input); Homo sapiens; OTHER
HUVEC, either untreated (control) or treated with TNF-α (10 ng/ml) for 4 h, were subjected to Cleavage Under Targets & Release Using Nuclease (CUT&RUN) using the CUT&RUN Assay Kit (#86652, Cell Signaling Technology) following the manufacturer's instructions. Cells from three independent biological replicates were pooled and crosslinked before being bound to Concanavalin A-coated magnetic beads. The cells were then permeabilized with Digitonin and incubated overnight at 4°C with H3K79me3 Rabbit mAb (1:50; #74073, Cell Signaling Technology). Chromatin-bound antibodies were targeted by pAG-MNase, and enzymatic digestion was initiated with calcium chloride. The resulting DNA fragments were incubated at 37°C to release enriched chromatin. Input DNA was prepared in parallel. DNA from both input and CUT&RUN-enriched samples was purified using the DNA Purification Buffers and Spin Columns Kit (#14209, Cell Signaling Technology). Input and H3K79me3-enriched DNA were processed using the NEBNext® Ultra™ II DNA Library Preparation Kit (#E7103, New England Biolabs) according to the manufacturer's protocol. Briefly, DNA samples were subjected to end repair and dA-tailing, followed by ligation with sequencing adapters. Adapter-ligated fragments were size-selected using SPRIselect magnetic beads (Beckman Coulter) to enrich for appropriate fragment lengths. Size-selected products were then amplified by limited-cycle PCR to incorporate unique index sequences and generate the final libraries. Library quality and quantity were assessed using a Bioanalyzer (Agilent Technologies) and Qubit Fluorometer (Thermo Fisher Scientific). High-throughput paired-end sequencing (2 x 150 bp) was performed on an Illumina NovaSeq 6000 platform (v1.5).