GSM7855670: ChIP_Seq-MYSTon_H4K12ac-S-1; Plasmodium falciparum; ChIP-Seq
To initiate cross-linking, 1% paraformaldehyde was immediately mixed with parasites and gently rotated for 10 min at 37 ℃. The cross-link reaction was quenched with 0.125 M glycine for 5 min on ice. RBCs were lysised with 0.15% saponin for 10- 15 min on ice and then subjected to three washes. Next, the parasites were incubated with 2 ml of Lysis Buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA pH8.0, 0.1mM EGTA pH8.0, 1mM DTT, 0.25% NP40, 1X protease inhibitors cocktail) for 30 min on ice, followed by dounce homogenization for 100 strokes. Following centrifugation, the precipitated nuclei were resuspended in 200 μl of SDS Lysis Buffer (containing 1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH8.0). The chromatin was sonicated using the Covaris M220 Sonicator with the following setting: 5% duty factor, 200 cycles per burst, and 75 W of peak incident power for 20 min to yield chromatin fragments at 200–500 bp in size. The chromatin fragments were diluted tenfold in a dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH8.0, 150 mM NaCl, 1X protease inhibitors cocktail) and co-incubated with Protein A/G magnetic beads (Pierce) for two hours. The supernatant was then co-incubated with Protein A/G magnetic beads and the corresponding antibody at 4 ℃ overnight. The antibodies used included 1 μg rabbit anti-GFP (Abcam, ab290), 1 μg rabbit IgG (Abcam), 3 μg anti-H3K9ac (Millipore, 07-352), anti-H3K9me3(Abcam, ab8898), and anti-H4K8ac(Abcam). The beads were subjected to several washes: once at 4℃ for 5 min with Low Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), High Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl) and LiCl Immune Complex Wash Buffer (0.25 M LiCl, 1% NP-40, 1% Deoxycholate, 1mM EDTA, 10 mM Tris-HCl pH 8.0), followed by two washes at room temperature for 5 min each with TE Buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0). The target protein-DNA complexes were eluted with 200 μL fresh Elution Buffer (1% SDS, 0.1 M NaHCO3). These complexes were then incubated at 45°C overnight, followed by incubation at 37°C for 30 min with RNase A and at 45°C for two hours with Proteinase K. ChIP-DNA was extracted using MinElute PCR purification kit (Qiagen, 28006). ChIP-DNA was end-repaired  (Epicentre No.ER81050), followed by the addition of 3' A base (NEB No.M0212L). Subsequently, adapter ligation was performed (NEB No.M2200L), and the libraries were size-selected and amplified using KAPA Biosystems KB2500. The amplification was carried out with a PCR program: including 1 min at 98 ℃, 12 cycles of 10 s at 98 ℃, and 1 min at 65 ℃, followed by a final extension of 5 min at 65 ℃.