title: GSM8686004: 3D7 H3K9Ac Schizont CUT&Tag rep1; Plasmodium falciparum; OTHER
library_name: GSM8686004
library_construction_protocol: For ChIP-seq, 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. For CUT&Tag-seq, 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 PfGCN5 CUT&Tag-seq, the bead-bound PfGCN5::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). For ChIP-seq, 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). For CUT&Tag-seq, 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.