title: GSM6368974: P. falciparum ORC1 ChIPseq 30 hpi (anti-HA); Plasmodium falciparum; ChIP-Seq
library_construction_protocol: Cells were fixed with 1% formaldehyde for 10 minutes at 37C. After quenching with 0.125 M glycine and saponin lysis, chromatin was sonicated using a M220 focused-ultrasonicator (Covaris). 5 ng of α-HA ChIP or input DNA were end-repaired with T4 DNA polymerase (NEB), Klenow DNA polymerase (NEB), and T4 Polynucleotide Kinase (NEB). The 3'ends of end-repaired DNA were extended with an A- overhang with 3' to 5' exonuclease-deficient Klenow DNA polymerase (NEB). The resulting fragments were ligated to Nextflex adapters (Bio Scientific) with the use of T4 DNA ligase (Promega). The libraries were amplified using an AT-rich optimized KAPA protocol using KAPA HiFi HotStart ready mix (KAPA Biosystems), NextFlex primer mix (Bio Scientific) with 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. The fragments originating from mono-nucleosomes + 125 bp NextFlex adapter were selected using 2% E-Gel Size Select agarose gels (Invitrogen) and amplified by PCR for 8 cycles using the above conditions. Libraries were purified and adapter dimers removed with Agencourt AMPure XP beads purification using a 1:1 library:beads ratio (Beckman Coulter). ChIP-seq libraries were sequenced on the Illumina NextSeq 500 system with a 20% phiX spike-in (Illumina) to generate 75 bp single-end reads (NextSeq 500/550 High Output v2 kit).