Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: The ChIP assay was performed as described in (Lopez-Rubio, 2012) with some modifications. Briefly, synchronized schizont stage parasite cultures were saponin-lysed until complete red blood cell lysis and formaldehyde-crosslinked. The isolated chromatin was sheared in SDS lysis buffer to obtain a fragment size of 100-150bp using an M220 focused-ultrasonicator (Covaris Inc.) and the following settings: 20% duty factor, 200 cycles per burst and total treatment time of 350s. After pre-clearing the chromatin with Magna ChIP protein A+G magnetic beads (Millipore), an input sample was collected and the rest of the chromatin was incubated overnight at 4C with 1μg of polyclonal anti-GFP antibody or, as control, the same amount of IgG. The immunoprecipitated chromatin was collected with Magna ChIP protein A+G magnetic beads (Millipore), extensively washed and eluted with elution buffer. The input and ChIP samples were reverse cross-linked overnight at 45C in the presence of 0.4M NaCl and purified. ChIP-qPCR was performed as described for ChIP-seq with few exceptions (details in supplemental experimental procedures of publication). Barcoded libraries for Illumina TruSeq single-end sequencing were constructed using NEBNext DNA library preparation reagents (NEB) by following the standard Illumina library preparation protocol. The libraries were PCR-amplified using Kappa HiFi (Kappa Biosystems) and purified using Agencourt AMPure XP beads (Beckman Coulter). The quality and percentage of adaptor-ligated material of the final sequencing libraries were determined by running them on an Agilent 2100 Bioanalyzer (Agilent Technologies). The concentration of each library was determined using a Quant-iT dsDNA Broad-Range Assay kit (Invitrogen).