HPIP controls osteoarthritis cartilage degeneration [ChIP-seq]

To assess the direct downstream targets of HPIP, a genome-wide ChIP-seq profiling was performed. The ChIP-seq data was examined by the quality evaluation. The results demonstrated that of significant ChIP-seq peaks for HPIP, 59% of them occurred at intronic and intergenic sites We found HPIP was closely correlated with the cartilage development. For ChIP assay, chondrocytes were fixed in 37% formaldehyde, pelleted, and resuspended in lysis buffer. The cells were sonicated and centrifuged to remove insoluble material. The supernatants were collected, and Pellet Protein G magnetic beads were added and incubated for 1 hour at 4°C with anti-HPIP (Proteintech, 1:50, 12102-1-AP). The chromatin was collected, purified, and de-crosslinked at 62°C for 2 h, followed by incubation at 95°C for 10 min. The DNA was isolated using the ChIP DNA Clean & Concentrator (Zymo Research Corp.) according to the manufacturer’s instructions. 100ng DNA fragments were used per ChIP. Sequencing libraries were prepared from collected HPIP ChIP and corresponding input DNA by blunting, A-tailing, adaptor ligation using NEXTFlex barcoded adapters (Bioo Scientific). Libraries were PCR-amplified for 16 cycles, fragments size of 300bp were processed using the Covaris Focused-ultrasonicators (Covaris, Inc. S220). Sequencing was performed using the Hi-Seq 2500 (Illumina) platform contributor: Novel Bioinformatics Co, Ltd