ATAC-seq of Human colorectal cancer cells: control blank
Cells or nuclei were first immobilized on concanavalin A-coated magnetic beads to facilitate subsequent reactions. The immobilized chromatin was incubated with a specific primary antibody targeting the protein of interest. After binding, a secondary antibody, coupled with Protein A-Tn5 transposase, was introduced. This enzyme-antibody complex simultaneously binds to the primary antibody and tagmentates the chromatin at the target sites, inserting sequencing adapters into the DNA. After tagmentation, the chromatin was released, and the DNA was purified. The adapter-ligated DNA fragments were then PCR-amplified to generate the sequencing library. Size selection was performed to enrich fragments within the desired size range, typically 200-600 bp, using AMPure XP beads (Beckman Coulter, Brea, CA, USA). The final libraries were assessed for quality and size distribution using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA) and quantified via real-time PCR. Sequencing was performed on the Illumina NovaSeq 6000 platform using paired-end technology (PE 150).