GSM3400938: AP2-G rings input, rep 1; Plasmodium falciparum; ChIP-Seq
ChIP was performed as previously described (PMID:28618269) with minor modifications. Shld1 was added to AP2-G-DD cultures at 24 hpi to stabilize AP2-G and nuclei were harvested 12 (committed schizonts), 36 (sexual rings), or 60 (stage I gametocytes) hours later.  Crosslinked nuclei were then resuspended in Covaris shearing buffer (0.1% SDS, 10 mM Tris pH 8.1, 1 mM EDTA) and sonicated using an M220 ultrasonicator (Covaris) and the following conditions: 5% duty cycle, 75 W peak incident power, 200 cycles per burst, treatment time 300 seconds.  The sonicated chromatin was diluted ten-fold with dilution buffer and precleared with Protein A/G magnetic beads (Pierce) for two hours. The pre-cleared material was then incubated with antibody and magnetic beads overnight, with a small volume of non-immunoprecipitated input material kept separately. For immunoprecipitations with AP2-G-DD, rat α HA (Roche 3F10) was used. The next day, beads were washed and the bound DNA eluted in elution buffer. Crosslinking was reversed by incubating overnight at 45°C and DNA was subsequently treated with RNase A at 37° for 30 minutes and Proteinase K at 45° for two hours. DNA was purified the Qiagen MinElute kit and quantified using the Qubit HS DNA assay. Barcoded libraries for Illumina TruSeq single-end sequencing were constructed using NEBNext DNA library Prep reagents (New England Biolabs) by following the standard Illumina library preparation protocol. The DNA was first end-repaired for 30 min at 20°C, purified using Agencourt AMPure XP beads (Beckman Coulter) and then dA-tailed for 30 min at 37°C, and purified using Ampure XP beads. NEXTflex Illumina DNA barcodes (diluted 1/10) were then ligated to the DNA fragments using T4 DNA ligase at 20°C for 15 min. The resulting DNA was then size selected for 250 bp inserts using Ampure XP beads PCR-amplified (12 cycles) using Kapa HiFi (Kapa biosystems) and purified using AMPure XP beads.
GEO Accession
GSM3400938