ChIP–seq on periosteal cells derived from the pre-invasive group using antibodies to acetylated histone H3 Lys27 (H3K27ac), H3K4me3 and HIF-1a
To test whether HIF-1a can directly induce Timp1 expression in periosteal cells at the pre-invasive stage of head and neck squamous cell carcinoma (HNSCC), we performed Chromatin immunoprecipitation followed by sequencing (ChIP–seq) on periosteal cells derived from the pre-invasive stage of a mouse HNSCC bone invasion model by using antibodies to acetylated histone H3 Lys27 (H3K27ac), H3K4me3 and HIF-1a. Overall design: Primary periosteal cells (Ter119–CD45–CD31–PDPN+PDGFRa+ cells) were isolated from the tumour-excised calvarial tissues from the pre-invasive stage of a mouse HNSCC bone invasion model. Chromatin immunoprecipitation on 100,000 periosteal cells was performed with CUT&RUN Assay Kit (CST, #86652) using antibodies to acetylated histone H3 Lys27 (H3K27ac) (CST,#8173s), H3K4me3 (CST,#9751) and HIF-1a(CST,#36169). Illumina sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (Cat No. 7645) according to the manufacturer's instructions. Barcoded libraries were run on the NovaSeq 6000 in a PE150 run, and an average of 40 million paired reads were generated per sample.