GSM7596960: ChIP-seq on PfSNF2L in a PfSNF2L+3xHA parasite line at 10hpi (hours post invasion); negative control IP; Plasmodium falciparum; ChIP-Seq
PfSNF2L 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). ChIP experiments were performed on PfSNF2L+HA parasites at four timepoints (10/22/34/46 hpi) in duplicate with an additional no-epitope control on WT 3D7 parasites. In brief, the protocol includes five steps: (1) Treated with 1% formaldehyde to crosslink the suspended PfSNF2L+HA parasite cultures at 37°C for 10 minutes; (2) Collected parasite nuclei using prechilled glass dounce homogenizer for 200 strokes per 10^9 rings and 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]) 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 PfSNF2L 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 P1 150 x 150 paired-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.