title: GSM4512249: PfAP2-G5_GFP_WT_D5_rep2_TF_ChIP-seq; Plasmodium falciparum; ChIP-Seq
library_construction_protocol: Synchronized PfAP2-G5-GFP::WT, PfAP2-G-GFP::WT, PfAP2-G-GFP::pfap2-g5_trun parasites at different stages were harvested and cross-linked immediately with 1% paraformaldehyde (Sigma) by rotating for 10 min at 37 ℃, then quenched with 0.125 M glycine for 5 min. The culture was resuspended with 1 x PBS and parasites were released from iRBC with 0.15% saponin for 10- 15 min on ice. The washed nuclei were isolated by incubation 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, 1 x protease inhibitors cocktail) for 30 min on ice, and followed by dounce homogenization for 100 strokes. Afterwards the nuclei were resuspended in 200 μl of SDS Lysis Buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH8.0) for sonication to generate 200–500 bp fragments in length. The sonicated chromatin was diluted ten-fold with dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH8.0, 150 mM NaCl, 1 x protease inhibitors cocktail) and precleared with Protein A/G magnetic beads (Pierce) for two hours. The precleared chromatin supernatant was incubated with 1 μg rabbit anti-GFP (Abcam, ab290) or rabbit IgG (Abcam) as control and Protein A/G magnetic beads at 4 ℃ overnight with a small volume of non-immunoprecipitated input material kept separately. After extensive washes, protein-DNA complexes were eluted with Elution Buffer (1% SDS, 0.1 M NaHCO3). Crosslinking was reversed by incubating overnight at 45°C and DNA was treated with RNase A at 37°C for 30 min and Proteinase K at 45°C for two hours. ChIP-DNA was extracted using MinElute PCR purification kit (Qiagen, 28006). ChIP-DNA was end-repaired (Epicentre No.ER81050), adding protruding 3' A base (NEB No.M0212L), adapter ligation(NEB No.M2200L), size selection and amplification of libraries(KAPA Biosystems, KB2500) with a PCR program: 1 min at 98 ℃, 12 cycles of 10 s at 98 ℃, 1 min at 65 ℃; finally extended 5 min at 65 ℃.
attributes.tag: GEO Accession
attributes.value: GSM4512249