GSM6636906: ChIP-seq of Plasmodium blood stages: R H3K9ac; Plasmodium falciparum; ChIP-Seq
Parasites were crosslinked with 1% paraformaldehyde. Crosslinked parasites were washed with 1 ml cold PBS containing protease inhibitors (cOmplete protease inhibitor cocktail) then incubated on ice with 1 ml nuclear extraction buffer [f.c.: 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 200 mM AEBSF, cOmplete protease inhibitor cocktail, Phos Stop phosphatase inhibitor in nuclease-free water]. After 35 min., NP-40/IGEPAL was added to a final concentration of 0.25% and the solution was homogenized by passing the mixture seven times through a 26 G needle. Extracted nuclei were pelleted by centrifugation at 2,100 × g for 20 min. at 4°C. Supernatant was removed and the pelleted nuclei were resuspended in 130 µl of cold shearing buffer [f.c.: 0.1% SDS, 1 mM EDTA, 10 mM Tris-HCl pH 7.5, cOmplete protease inhibitor cocktail, Phos Stop phosphatase inhibitor in nuclease-free water] and transferred to a microTUBE. Chromatin was fragmented with an M220 sonicator (Covaris) with the following settings: 5% duty cycle, 75 W peak incident power, 200 cycles per burst at 6°C for 300 sec. Fragmented chromatin was transferred to a pre-lubricated, low binding 1.5 ml tube. The microTUBE was washed with 70 µl of shearing buffer after which the solution was combined with the fragmented chromatin for a total volume of 200 µl. For each developmental stage (schizont, merozoite, ring, and hepatic merozoite) 30 µl of sheared chromatin was collected at this point for use as an input sample and stored at -70°C until decrosslinking. Chromatin was diluted 1:1 in ChIP dilution buffer [f.c.: 30 mM Tris-HCl pH 8.0, 3 mM EDTA, 0.1% SDS, 300 mM NaCl, Triton X-100, supplemented with cOmplete protease inhibitor cocktail and Phos Stop phosphatase inhibitor in nuclease-free water] then precleared by incubation with protein A agarose/salmon sperm DNA beads (50 µl beads per 1 ml sheared chromatin) for 1 hour at 4°C with agitation to reduce non-specific background. Following the incubation, the beads were pelleted by centrifugation at 100 × g for 1 min. at RT. The supernatant containing the chromatin was moved to a new low-binding tube and incubated overnight at 4°C with agitation with 2 µg of antibody against the histone PTM of interest. Protein A agarose/salmon sperm DNA beads (25 µl beads per 500 µl sonicated chromatin) were washed with 500 µl ChIP dilution buffer and blocked with 1 mg/ml BSA for 1 hour at 4°C with agitation to reduce non-specific binding. Agarose beads were washed 3 times with 500 µl ChIP dilution buffer, then added to the chromatin suspension and incubated for 1 hour at 4°C with agitation. Agarose beads were pelleted by centrifugation at 100 × g for 1 min. at RT, and the supernatant was discarded. Beads were washed twice at 4°C for 15 min. with agitation 1 ml high salt wash buffer [f.c.: 1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 nM NaCl in nuclease-free water], 1 ml low salt wash buffer [f.c.: 1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl, in nuclease-free water], and 1 ml lithium chloride wash buffer [f.c.: 0.25 M LiCl, 1% NP-40/IGEPAL, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0 in nuclease-free water]. Beads were then washed twice for 15 min. with TE buffer [f.c.: 10 mM Tris-HCl pH 8.0, 1 mM EDTA in nuclease-free water] at RT with agitation. Chromatin bound to agarose beads was eluted by incubating with 125 µl elution buffer [f.c.: 1% SDS, 0.1 M NaHCO3 in nuclease-free water] for 15 min. at RT with agitation. The elution was repeated, and fractions combined for a total of 250 µl. Samples were reverse crosslinked by adding NaCl to a final concentration of 0.5 M and incubating overnight at 45°C. RNase A was then added to a final concentration of 0.6 μg/ml and incubated at 37°C for 30 min. Samples were then incubated with EDTA (f.c.: 1 mM), Tris-HCl pH 7.5 (f.c.:4 mM), and proteinase K (0.8 U) for 2 hours at 45°C. DNA was isolated using 2 volumes sparQ beads according to manufacturer's instructions. ChIP DNA was stored at -20°C until library preparation. Input samples were incubated with 0.6 μg RNase A per µl for 30 min. at 37°C, then incubated with 0.8 U proteinase K overnight at 45°C. DNA was isolated using 2 volumes sparQ beads according to manufacturer's instructions and stored at -20°C until library preparation. Libraries from ChIP samples were prepared using the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB #E6040) or the NEBNext Ultra II DNA library prep kit for Illumina (NEB #E7645) according to manufacturer's instructions with the following modification. Libraries were amplified using i7 index primers and i5 universal primer (NEBNext multiplex oligos for Illumina index primers set 1 and 2, NEB #E7335 and #E7500) with KAPA HiFi hot start ready mix with the following program: initial denaturation at 98°C for 45 sec.; 15 cycles of (98°C for 15 sec., 55°C for 30 sec., 62°C for 30 sec.); and final extension at 62°C for 5 min. Libraries were purified using 0.9 volumes sparQ beads according to manufacturer's instructions. Libraires were sequenced with the Illumina HiSeq 2500 or Illumina NextSeq500 to generate 50 bp single-end reads or 75 bp paired-end reads, respectively.