GSM7664033: ChIP-seq on PfMORC in a PfMORC+3xHA parasite line at 30hpi (hours post invasion); replicate 1 input; Plasmodium falciparum 3D7; ChIP-Seq
PfMORC ChIP-seq was performed similarly to previously published ChIP-seq experiments in Plasmodium falciparum (Josling et al. 2020, Singh et al. 2021, Russell et al. 2022). Five samples in total were collected: biological duplicates using the PfMORC+HA parasite line at the trophozoite stage [30 hpi], biological duplicates using the PfMORC+HA parasite line at the schizont stage [40 hpi], and a single replicate using the PfMORC+GFP parasite line at the schizont stage [40 hpi]. In brief, the protocol includes five steps: (1) Treated with 1% formaldehyde to crosslink the suspended PfMORC+HA (or PfMORC+GFP) parasite cultures (at least 10^8 trophozoite- or schizont-stage parasites synchronized with 10% sorbitol more than one cycle prior) at 37°C for 10 minutes; (2) Collected parasite nuclei using prechilled glass dounce homogenizer for 100 strokes per 10^9 trophozoites/schizonts; (3) Lysed parasite nuclei and mechanically sheared chromatin until sufficiently sheared using Covaris Focus-Ultrasonicator M220 (5% duty cycle, 75 W peak incident power, 200 cycles per burst, 7°C, for 5 minutes). (4) We collected 10% of each sample for the nonimmunoprecipitated “input” control and then immunoprecipitated the remaining 90% of each sample. The remaining 90% of each sample was immunoprecipitated with 1:1000 anti-HA antibody (0.5 mg/mL Roche Rat Anti-HA High Affinity [11867423001]) or 1:1000 anti-GFP antibody (0.1 mg/mL Abcam Rabbit Anti-GFP [Ab290]) overnight at 4°C with rotation. (5) DNA was purified after reverse crosslinking using a MinElute column (Qiagen) as directed and quantified by a Qubit fluorometer (Invitrogen). The PfMORC ChIP-seq libraries were performed similarly to previously published ChIP-seq experiments in Plasmodium falciparum (Josling et al. 2020, Singh et al. 2021, Russell et al. 2022). DNA sequencing libraries were prepared for high-throughput Illumina sequencing on the NextSeq 2000 with the 150 x 150 single-end mode. The library prep underwent 12 rounds of amplification using KAPA HiFi polymerase. Completed libraries were quantified using the Qubit fluorometer (Invitrogen) for high-sensitivity DNA and library sequence length by the Agilent TapeStation 4150.