Effect of MS-275 and CXD101 on open chromatin regions of M-MDSCs [ATAC-seq]

To elucidate the molecular mechanisms of HDAC1-3 inhibitors in inhibiting M-MDSCs, ATAC-seq analysis of M-MDSCs isolated from Hepa 1-6 tumor-bearing mice treated with MS-275 or CXD101 were performed. M-MDSCs (CD11b+Ly-6ChiLy-6G-) were sorted by JSAN (KS-Techno, Japan) from tumor isolated. ATAC-seq libraries were prepared using ATAC-seq Kit (Active Motif, USA) according to the manufacturer’s protocol. Briefly, 1 × 105 M-MDSCs were lysed with ATAC lysis buffer and nuclei were isolated by centrifugation at 500 × g, 4°C for 10 min. Nuclei were then suspended in 50 µL of Tagmentation Master Mix and incubated at 37°C for 30 min with shaking. DNA was purified using DNA purification column and PCR amplification was performed for 10 cycles to introduce sequencing adapters to open chromatin regions. Library DNA was finally purified using Solid Phase Reversible Immobilization beads. Molecular weight was checked and libraries were quantified using Bioanalyzer and submitted for NGS analysis.