GSM9105646: sgTRAPPC4; Homo sapiens; OTHER
For CUT&Tag experiments targeting FOS, LIU-LSC-1 cells were harvested and washed with wash buffer. Intact nuclei were isolated using digitonin-based permeabilization and immobilized on concanavalin A-coated magnetic beads. Nuclei-bound cells were incubated overnight at 4°C with a primary antibody against FOS (1:50 dilution), followed by a secondary antibody incubation to amplify the signal. Protein A-Tn5 transposome was then added to bind the antibody complexes. After washing, tagmentation was performed by adding Mg²⁺ to activate Tn5, simultaneously fragmenting DNA and incorporating sequencing adapters. DNA was then extracted by reverse crosslinking, followed by purification using AMPure XP beads. CUT&Tag libraries were constructed following the published protocol by Kaya-Okur et al. (2019, Nature Communications). Briefly, concanavalin A-coated magnetic beads were used to immobilize the nuclei. After digitonin permeabilization, samples were incubated overnight at 4°C with a primary antibody against the target protein (FOS), followed by incubation with a secondary antibody. Protein A-Tn5 fusion enzyme loaded with sequencing adapters was then added to bind the antibody complex. After tagmentation at 37°C for 1 hour, DNA was released by adding SDS and Proteinase K, followed by overnight incubation at 55°C. DNA fragments were purified using AMPure XP beads. Library amplification was performed by PCR using indexed primers, and libraries were size-selected (typically 300–700 bp) using magnetic beads. The final libraries were quantified and sequenced on the Illumina PE150 platform.