ChIP-seq detection of transcription factor p65-enriched DNA fragments in rheumatoid arthritis patient-derived synovial fibroblasts

Synovial fibroblasts were isolated from synovial tissues of rheumatoid arthritis patients, passaged, and purified, and identified by flow cytometry. Cells from passages 3 to 8 were used for experiments. Synovial fibroblasts were stimulated with TNF for 24 h. Synovial fibroblasts (1×10⁷) were PBS-washed, cross-linked in 1% formaldehyde (10 min, room temperature), and quenched with 0.125 M glycine (5 min). After centrifugation and PBS washes, cells were lysed in ice-cold buffer (0.5% NP-40, 10 mM HEPES pH 7.5, 0.1 mM EDTA, protease inhibitors) for 5 min. Nuclei were pelleted (2000×g, 10 min, 4°C), and chromatin was sonicated to 100-500 bp fragments. Sonicated chromatin was divided: 10% retained as "input," 80% immunoprecipitated with anti-p65 antibody (CST #8242), and 10% incubated with rabbit IgG (Abcam ab171870) as "IgG" control. IP complexes were sequentially washed, eluted, and de-crosslinked. DNA from input and IP fractions was phenol-chloroform-extracted, quantified (Qubit 3), and quality-checked by agarose gel electrophoresis (chromatin fragmentation) and Western blot (IP efficiency). Sequencing libraries were prepared using VAHTS Universal DNA Library Prep Kit (Vazyme ND607), size-selected (200-500 bp), and sequenced on an Illumina Novaseq X Plus (PE150 mode).