GSM5462678: 3D7_AP2-06A-GFP_S_AP2-06A_ChIP-seq, Rep1; Plasmodium falciparum; ChIP-Seq
Synchronized parasites were harvested at the schizont stage (40-45 hpi) and crosslinked immediately with 1% paraformaldehyde (Sigma) by rotating for 10 minutes at 37°C, which was then quenched with 0.125 M glycine for 5 minutes on ice. Parasite nuclei released from infected red blood cells were sheared for 20-30 minutes using an M220 sonicator (Covaris) at 5% duty cycle, 200 cycles per burst, and 75 W of peak incident power to generate 100-500 bp fragments in length. 20 µL of the sheared chromatin was reserved as the input control. Chromatin was subsequently immunoprecipitated overnight at 4°C using 0.5 µg of antibodies against GFP (Abcam, ab290) and protein A/G magnetic beads (ThermoFisher Scientific, 26162). After extensive washes, the immunoprecipitated materials were eluted with elution buffer. Then crosslinking was reversed by overnight incubation at 45°C, followed by treatments of RNase A at 37°C for 30 minutes and Proteinase K at 45°C for two hours. The ChIPped DNA was finally purified according to the MinElute PCR purification kit (Qiagen, 28006) instructions. 1.5 ng of ChIP-DNA was subjected to end-repair (Epicentre, ER81050), 3' adenylation (NEB, M0212L), and adapter ligation (NEB, M2200L). Then after purification with Agencourt AMPure XP beads (Beckman Coulter), libraries were amplified using the KAPA HiFi PCR Kit (KAPA Biosystems, KB2500) under the following conditions: 1 minute of initial denaturation at 98°C, 12 cycles of 10 seconds at 98°C and 1 minute at 65°C, and 5 minutes of final extension at 65°C.