Instrument: Illumina HiSeq 2000
Strategy: ATAC-seq
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: Cultures were tightly synchronized to a defined 5 h age window by purification of parasites at the schizont stage using Percoll gradients (63% Percoll) followed by sorbitol lysis 5 h later, which eliminates erythrocytes infected with late asexual stages (trophozoites and schizonts). Parasites were collected at the following times post invasion: 10-15 hpi (early ring stage), 20-25 hpi (late ring stage), 30-35 hpi (trophozoite stage) and 40-45 hpi (schizont stage). Genomic DNA was isolated by proteinase K treatment (0.2 mg/ml, 55°C for 3 hr and 95°C for 25 min), then RNase treatment (1.25 mg/ml, 37°C for 30 min). Genomic DNA was purified from schizonts by phenol/chloroform/isoamyl alcohol (25:24:1, pH 8.0) extraction, with an additional chloroform extraction, and precipitated with ethanol and NaOAc (3mM, pH 5.5), and resuspended in TE (10mM Tris-HCl pH7.5, 1mM EDTA). 10ng of gDNA used for a standard ATAC-seq protocol as described by the Nextera library preparation protocol by Illumina (5 min at 55º C). ATAC-seq protocol was performed using 10 million cells for early ring, late ring and throphozoite cell stages and 1 million cells for schizonts that were obtained after saponin RBC lysis. Parasite cells were resuspended in lysis buffer in order to permeabilize membranes. The nuclei pellet were immediately resuspended in the transposition reaction mix (25 μl of 2x TD Buffer, 1.25 μl of Tn5 Transposase and 23.75 μl of nuclease-free water), and incubated for 30 min at 37ºC. The samples were purified using the Qiagen MiniElute Kit. Following purification, library amplification was carried out with 2X KAPA HiFi mix and 1.25 μM of Nextera primers. Optimal cycle number was determined using qPCR. ATAC-seq libraries were sequenced using an Illumina HiSeq2000 sequencer to obtain 20-40M of 2x50 bp paired-end sequencing reads.