Chip grade protein g magnetic beads 9006

A total of 4 × 10⁶ cells were fixed with 1% formaldehyde, lysed, and sonicated (Branson Sonicator; Branson Ultrasonics, Danbury, CT, USA) leading to a DNA average size of 200 bp. 5 µg of antibodies anti-STAT3 #4904 (Cell Signaling), H3K4me3 (ab8580, Abcam, Cambridge, UK) H3K27Ac (ab177178, Abcam), H3K27me3 (ab6002, Abcam), or control IgG #2729 (Cell Signaling) were added to the precleared sample and incubated overnight at 4  °C. The complexes were purified using CHIP-grade protein-G magnetic beads #9006 (Cell Signaling), followed by elution from the beads and reverse cross-linking. DNA was purified using PCR purification columns (Qiagen) and target control genes (TNFRSF8 and GAPDH were amplified by real-time quantitative PCR using SYBR Green (Bio-Rad, #1725272). The oligonucleotide primer pairs are reported in Table S5. Raw ChIP-Seq samples were aligned using bwa aligner. Peak calling was performed using Model-based Analysis of ChIP-Seq (MACS Version 2.1.1, [72 (link)]), an open source computational algorithm for identifying genome-wide protein-DNA interaction from ChIP-Seq data (https://github.com/taoliu/MACS). Only peaks with FDR < 0.05 were called as significantly enriched. Signal tracks were created using MACS2 bdgcmp command. Peak enrichment relative to IgG was calculated in bedgraph format and then converted to bigwig using UCSC toolkit.

MB‐3 (M2449), complete Freund's adjuvant (CFA) (F5881), and LPS (E.coli 0111:B4) were purchased from Sigma–Aldrich. Chicken Type II Collagen (20088) was from Chondrex. ChIP‐grade protein G magnetic beads (9006) and cell lysis buffer (9803) were from Cell Signaling Technology (CST). Nigericin (tlrl‐nig), ATP (tlrl‐atpl), and MSU (tlrl‐msu) were from InvivoGen. Mouse IL‐1β, TNF‐α, and IL‐6 ELISA kits were from R&D Systems. Antibodies against KAT2A (3305), IL‐1β (12242), NRF2 (12721), IRG1 (19857), Acetyl‐Histone H3 Lys9 (9649), and β‐Actin (3700) were from CST. Antibodies against NLRP3 (AG‐20B‐0014B), caspase 1 (AG‐20B‐0042B), and ASC (AG‐25B‐0006PF) were from AdipoGen Life Sciences. Antibodies against CD45‐BV605 (2D1), CD4‐BV421 (GK1.5), CD8a‐BV510 (53‐6.7), CD11b‐BV510 (M1/70), F4/80‐FITC (BM8), CD44‐APC (IM7), CD62L‐PE (MEL‐14), PD‐1‐APC (29F.1A12), CXCR5‐PE (J252D4), IFN‐γ‐APC (XMG1.2), and IL‐17a‐PE (TC11‐18H10.1) were from Biolegend. Antibody against FoxP3‐PE (FJK‐16s) was obtained from eBioscience.