GSM3355027: ChIPseq_SMC3_Rep1; Plasmodium falciparum; ChIP-Seq
Synchronized parasite cultures were collected and subsequently lysed by incubating in 0.15% saponin for 10 min on ice. Parasites were centrifuged at 3,234 x g for 10 min at 4°C, and washed three times with PBS. For each wash, parasites were resuspended in cold PBS and centrifuged for 10 min at 3,234 x g at 4°C. Subsequently, parasites were crosslinked for 10 min with 1% formaldehyde in PBS at 37°C.  Glycine was added to a final concentration of 0.125 M to quench the crosslinking reaction, and incubated for 5 min at 37°C. Parasites were centrifuged for 5 min at 2,500 x g at 4°C, washed twice with cold PBS and stored at -80°C. For chromatin immunoprecipitation, parasites were first incubated on ice in nuclear extraction buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), EDTA-free protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche)). After 30 min, Igepal CA-360 (Sigma-Aldrich) was added to a final concentration of 0.25% and the parasites were lysed by passing the suspension through a 26 G ½ inch needle seven times. Parasite nuclei were centrifuged at 4°C for 20 min at 2,500 x g.  Parasite nuclei were resuspended in shearing buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris HCl pH 7.5, EDTA-free protease inhibitor cocktail, and phosphatase inhibitor cocktail). Chromatin was fragmented using the Covaris Ultra Sonicator (S220) for 10 min with the following settings; 5% duty cycle, 140 intensity peak incident power, 200 cycles per burst). To remove insoluble material, samples were centrifuged for 10 min at 17,000 x g at 4°C. Fragmented chromatin was diluted 1:1 in ChIP dilution buffer (30 mM Tris-HCl pH 8, 3 mM EDTA, 0.1% SDS, 300 mM NaCl, 1.8% Triton X-100, EDTA-free protease inhibitor cocktail and phosphatase inhibitor cocktail). Samples were precleared with Protein A Agarose beads to reduce non-specific background and incubated overnight at 4°C with 2 μg of custom anti-SMC3 antibody (Thermo Fisher Scientific). A sample with no antibody was also incubated overnight at 4°C to be used as the negative control. Antibody-protein complexes were recovered using Protein A Agarose beads, followed by extensive washes with low salt immune complex wash buffer, high salt immune complex was buffer, LiCl immune complex wash buffer and TE buffer. Chromatin was eluted from the beads by incubating twice with freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3) for 15 min at RT. Samples were reverse crosslinked overnight at 45°C by adding NaCl to a final concentration of 0.5 M. RNase A (Life Technologies) was added to the samples and incubated for 30 min at 37°C followed by a 2 h incubation at 45°C with the addition of EDTA (final concentration 8 mM), Tris-HCl pH 7 (final concentration 33 mM) and proteinase K (final concentration 66 μg/mL; New England Biolabs). DNA was extracted by phenol:chloroform:isoamylalcohol and ethanol precipitation. Extracted DNA was purified using 1.8X Agencourt AMPure XP Beads (Beckman Coulter). Libraries from the ChIP samples were prepared using the KAPA Library Preparation Kit (KAPA Biosystems).  Libraries were amplified for a total of 12 PCR cycles (12 cycles of [15 s at 98°C, 30 s at 55°C, 30 s at 62°C]) using the KAPA HiFi HotStart Ready Mix (KAPA Biosystems).  Libraries were sequenced with a NextSeq500 DNA sequencer (Illumina)