title: GSM8942325: PF3D7, WR+/-, ChIP-Input; Plasmodium falciparum; ChIP-Seq
library_name: GSM8942325
library_construction_protocol: RBC culture pellets were crosslinked with 1% formaldehyde for 10 min at 37ºC followed by quenching with 0.125 M glycine. Parasites were isolated by RBC lysis in 0.05% saponin and preserved at -80°C . Nuclei were obtained by lysing the parasites in cytoplasmic lysis buffer. Chromatin was sheared to 100-600 bp fragment size range by sonication for 20 cycles of 30 sec ON/30 sec OFF in sonication buffer. ChIPs were performed under rotation overnight at 4°C in incubation buffer containing sonicated chromatin and either rabbit α-PfHP1 or  rabbit  IgG negative control antibodies in the presence of 10 μl Dynabeads Protein A and Dynabeads Protein G. Magnetic beads were washed six times and immunoprecipitated chromatin was eluted with 1% SDS, 0.1 M NaHCO3. After de-crosslinking at 45°C in a shaking heat block overnight, DNA fragments were purified using QIAquick MinElute PCR columns. The purified DNA fragments were end-repaired, extended with 3′ A-overhangs and ligated to barcoded NextFlex adapters. Libraries were amplified using KAPA HiFi HotStart ready mix, NextFlex primer mix and the following PCR program: 98°C for 2 min; four cycles of 98°C for 20 sec, 62°C for 3 min; 62°C for 5 min. Amplified libraries were size-selected for 225-325 bp (mono-nucleosomes + 125 bp NextFlex adapter) using 2% E-Gel Size Select agarose gels and amplified by PCR for ten cycles using the same PCR program. Libraries were purified by Agencourt AMPure XP beads purification using a 1:1 library:beads ratio.