Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Parasites were resuspended in ice cold SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1) supplemented with PMSF (100μM/ml) and protease inhibitor cocktail for 10 minutes on ice. Parasite lysate was then sonicated to generate the sheared chromatin of ~500bp and centrifuged at 1000g for 15 minutes. After the removal of debris from the lysate, the lysate was further diluted 10 times in ChIP dilution buffer (0.01% SDS, 0.1% Triton-X 100, 1.2mM EDTA, 16.7mM Tris, pH 8.1, and 150mM NaCl). Subsequently, the parasite lysates were precleared using protein A sepharose beads at 4°C for 1 hour. Cleared chromatins were incubated with pre-immune sera and immune GCN5 sera for overnight at 4°C on rotating wheel. The paired-end sequencing library was prepared using Illumina TruSeq Nano DNA LT Library Preparation Kit. 200ng of each DNA was fragmented by Covaris S2. Covaris shearing generates DNA fragments with 3' or 5' overhangs. The fragments were then subjected to end-repair. This process converts the overhangs resulting from fragmentation into blunt ends using End Repair Mix. The 3' to 5' Exonuclease activity of this mix removes the 3' overhangs and the 5' to 3' polymerase activity fills in the 5' overhangs. A single "A" nucleotide is added to the 3' ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. A corresponding single "T" nucleotide on the 3' end of the adapter provides a complementary overhang for ligating the adapter to the fragment. This strategy ensures a low rate of chimera (concatenated template) formation. Indexing adapters were ligated to the ends of the DNA fragments, preparing them for hybridization onto a flow cell. The ligated products were purified using Ampure XP beads. The product was PCR amplified as described in the kit protocol.