Chromatin landscape of H3K9ac and its modifier GCN5 in the malaria parasite

This study is to analyze the chromatin landscape of the active mark H3K9ac, an active mark catalyzed by GCN5, and GCN5 in the malaria parasite Plasmodium falciparum at the schizont stage by both ChIP-seq and CUT&Tag-seq for H3K9ac and CUT&Tag-seq for GCN5. In this study, wild-type 3D7 parasites at the schizont stage were harvested for H3K9ac ChIP-seq and CUT&Tag-seq for H3K9ac and GCN5. For H3K9ac ChIP-seq was conducted by the standard methods. Briefly, Synchronized WT 3D7 parasites at the schizont stage were harvested and crosslinked with 1% paraformaldehyde and then neutralized by glycine (0.125 M). The fixed iRBCs were lysed with saponin (0.06% final concentration) and parasites were treated with a lysis buffer (10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 10 mM Hepes, pH 7.9, 1 × protease inhibitor) and then were homogenized using a douncer to free the nuclei. Pelleted nuclei were sonicated in a shearing buffer (0.1% SDS, 5 mM EDTA, 50 mM Tris-HCl, pH 8.1, 1× protease inhibitor) using a rod bioruptor (Microson ultrasonic cell disruptor, Misonix, Inc. USA) at high power for 20 cycles of 30 sec ON/30 sec OFF, resulting in sheared chromatin of approximately 100–1000 bps. Fifty μl of input samples were set aside, and the remaining chromatin was diluted in an incubation buffer (0.01% SDS, 1.5% Triton X-100, 0.5 mM EDTA, 200 mM NaCl, 5 mM Tris-HCl, pH 8.1). The chromatin (75 μl/400 ng) was incubated with rabbit anti-H3K9ac (Diagenode pAb-004-050) followed by the addition of 20 μl of agarose beads. Beads were then washed with the following: buffer 1 (0.1% SDS, 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris HCl, pH 8.1); buffer 2 (0.1% SDS, 1% Triton X-100, 500 mM NaCl, 2 mM EDTA, 20 mM Tris HCl, pH 8.1), buffer 3 (250 mM LiCl, 1% NP-40, 1% Na-deoxycholate, 1 mM EDTA, 10 mM Tris HCl, pH 8.1) and finally twice with buffer 4 (10 mM EDTA, 10 mM Tris HCl, pH 8). The immunoprecipitated (IPed) chromatin was eluted with the elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature. The eluted chromatin and input samples were reverse crosslinked in 10% SDS, 1MNaHCO3, 5MNaCl, and 10% Triton X-100 at 45˚C overnight while shaking and purified by the phenol:chloroform method. The immunoprecipitated gDNA (~20 ng/sample) was then used to generate libraries using the KAPA Hyper prep kit for the Illumina sequencing platform according to the manufacturer's protocol (KAPA Biosystems). Pooled libraries were sequenced on the Illumina NextSeq 550 instrument using 150 bp paired-end sequencing and dual indexing. CUT&Tag-seq for H3K9ac and GCN5 was conducted by the establshed methods (PMC6488672) with some modifications. Briefly, parasites at the schizont stage were harvested and fixed with 1% formaldehyde followed by saponin lysis. The parasite pellet was suspended in a nuclear extraction buffer. Ten μl of Activated Concanavalin A-coated magnetic beads (EpiCypher # 21-1401) were added to each sample (~0.5 × 106 nuclei). For H3K9ac CUT&Tag-seq, the bead-bound WT parasite nuclei were resuspended in 50 μl Antibody150 buffer containing 0.5 μg of rabbit anti-H3K9ac (Diagenode pAb-004-050) as the primary antibody and rabbit IgG (Cell Signaling Technology #2729S) serving as a control. For GCN5 CUT&Tag-seq, the bead-bound GCN5::GFP nuclei were incubated with 0.5 μg of goat anti-GFP (Novus biologicals NB100-1770) while WT 3D7 nuclei were used as control. Following primary antibody incubation, nuclei were incubated with the secondary antibodies, donkey anti-goat IgG (Abcam ab6885). The nuclei were resuspened in Digitonin300 Buffer containing CUTANA pAG-Tn5 (EpiCypher #15-1017). Next, the nuclei were resuspended in Tagmentation buffer (10 mM MgCl2 in Digitonin300) at 37 °C for 1 h followed by washing with 50 µl TAPS buffer, quenching tagmentation with 5 µL SDS release buffer, incubating at 58ºC to release tagmented chromatin into the solution, and quenching SDS by adding 0.67% Triton-X. PCR with appropriate barcoded primers was performed using the CUTANA High Fidelity 2x PCR Master Mix (EpiCypher #15-1018) according to the manufacturer's recommendations. Amplified DNA libraries were captured by Kapa pure beads (Roche #KK8001) according to the manufacturer's recommendations, and 150 bp paired-end sequencing was performed on the NextSeq 550 platform.