Plasmodium falciparum MNase ChIP-Seq erythrocytic cycle 36h
Parasites were extracted from erythrocytes by saponin-lysis and crosslinked for 10 min at RT using 1% formaldehyde. Nuclei were isolated and permeabilized by mild sonication for 4 cycles of (15 s ON and 45 s OFF) at high intensity using a Bioruptor UCD-200 (Diagenode, Denville, NJ). Chromatin was digested by MNase (50 U) for 5 min at 37°C. Anti-histone H3 (Abcam ab1791) immunoprecipitations were performed using the Chromatin Immunoprecipitation Assay Kit (Millipore, Billerica, MA) according to the manufacturer's protocol. Samples were decrosslinked by O/N incubation at 45°C, followed by RNase A treatment (30 min at 37°C), proteinase K treatment (2 h at 45°C) and DNA purification. Sequence libraries were prepared using the NEBNext ChIP-Seq Library Preparation kit (NEB) according to the manufacturer’s instructions, with the exception of the use of the KAPA HiFi Hotstart Ready Mix for the amplification of the libraries. Libraries were amplified for a total of 11 PCR cycles (3 cycles of [15s at 98°C, 30s at 55°C, 30s at 62°C] followed by 8 cycles of [15s at 98°C, 30s at 63°C, 30s at 72°C]).