ChIP-seq analysis of porcine alveolar macrophages infected with T. gondii RH

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite that can infect almost all warm-blooded animals, causing serious public health problems. Lysine crotonylation (Kcr) is a newly discovered posttranslational modification (PTM), which has been proved that is relevant to procreation regulation, active transcription and cell signaling pathway. However, the biological functions of crotonylation have not yet been reported in macrophages infected with T. gondii. In our study, we performed a ChIP-seq analysis of porcine alveolar macrophages infected with T. gondii RH to explore the relationship of histone Kcr with T. gondii infection. Overall design: Porcine alveolar macrophages (3D4/21 cells) were infected with tachyzoites of T. gondii type ? strain RH?hxgprt (MOI=5) propagated in human foreskin fibroblast cells (obtained from ATCC, USA) in DMEM supplemented with 2% FBS at 37? with 5% CO2 or mock infected with an equal amount of phosphate-buffered saline (PBS, pH 7.4). After 24h post-infection, all samples were harvested and stored at -80 ? for subsequent protein and chromatin extraction. 3D4/21 cells were cross-linked with 1% formaldehyde for ran chromatin extraction. After sonication, fragmented DNA was obtained and then incubated with antibody (anti-H2BK12cr)-coated beads for 12h at 4?. After extensive washing, 20 µL 5M Nacl was added into immunoprecipitated chromatin to de-cross-linked overnight, and the concentration and purify of de-cross-linked product was measured for subsequent sequencing.