title: GSM3903870: RUF6-OE_HM-H3K9Ac_R_rep1_ChIP-seq; Plasmodium falciparum; ChIP-Seq
library_construction_protocol: Ring-stage (10 hr post invasion ±5 hr) synchronized 3D7-G7 and PfRrp6-DD-1C lines were used in ChIP assay. Parasites culture (1x109 rings) was harvested and cross-linked immediately with 1% paraformaldehyde with rotation for 10 min at 37℃, and quenched with 0.125 M glycine for 5 min on ice. The fixed culture was washed twice with cold 1xPBS, and the parasites were released from iRBC with 0.15% saponin for 15min on ice. After washed with cold 1xPBS, nuclei were isolated by incubation with 2 ml cold 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, and followed by dounce homogenization for 200 strokes. After centrifuge at 10,000 rpm for 10 min at 4 ℃, the nuclei was resuspended in 150μl SDS Lysis Buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH8.0) for sonication. Chromatin was sheared into 200-500 bp fragments with Bioruptor (BioruptorTM UCD-200) of highest power for 4x4 min at 30 sec intervals. The resulting chromatin was diluted 1:10 in ChIP 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). Pre-clear the chromatin solution with 50 μl/ml of salmon sperm DNA/protein A/G agarose slurry for 2h at 4℃ with agitation. After centrifugation, per 250μl chromatin supernatant was incubated with 3μg of Rabbit polyclonal H3K9me3 (abcam, ab8898), 3μg of Rabbit polyclonal H3K9Ac (Millipore, 07-352) and  3μg of Rabbit polyclonal HP1 for each IP overnight at 4℃ and rabbit IgG (Sigma) as control. To collect immune complexes, the sample was incubated with 50μl of Salmon sperm DNA/protein A/G slurry for 2h at 4℃ with agitation. After extensive washes, immunoprecipitated chromatin was eluted with ChIP Elution Buffer (1% SDS, 0.1 M NaHCO3), and reverse crosslinked at 65℃ for 16 h in the presence of 200 mM NaCl, then added RNase A at 37℃ for 30min and incubated with the proteinase K at 45℃ for 2h. DNA was purified by Phenol/chloroform/isoamylic alcohol extraction for ChIP-seq. To prepare sequencing libraries, 10 ng of input DNA, 3-5ng IPed DNA were end repaired, extended with 3′ A-overhangs and ligated to barcoded NextFlex adapters (Bio Scientific). Libraries were amplified using an optimized KAPA protocol using KAPA HiFi HotStart ready mix (KAPA Biosystems) and the following PCR program: 98°C for 3 min followed by 14 cycles of 98°C for 20 sec, 65°C for 30 s; 68°C for 30s; finally extended 68°C for 5min. Amplified libraries were size-selected for 300-600 bp using 2% agarose gels. ChIP-seq libraries were sequenced on the HiSeq X Ten machine.
attributes.tag: GEO Accession
attributes.value: GSM3903870