Hi-C WA1
100 ml in vitro culture of B. duncani WA1 in A+ human RBCs was grown to a parasitemia of 15%. The culture was centrifuged, and the parasite pellet was crosslinked in 1.25% formaldehyde for 25 min at 37C. Crosslinking was quenched in a final concentration of 150 mM glycine for 15 minutes at 37C followed by a 15-minute incubation at 4C. Parasite pellets were then resuspended in lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 2 mM 4-(2- aminoethyl) benzenesulfonyl fluoride HCl (AEBSF), 0.25% Igepal CA-360 (v/v), and EDTA-free protease inhibitor cocktail (Roche)) and incubated for 30 min on ice. Nuclei were isolated after homogenization by 15 needle passages. In situ Hi-C protocol was performed as described by Rao and colleagues (45). Briefly, nuclei were permeabilized using 0.5% SDS. DNA was digested with 100 units of Mbol (NEB), the ends of restriction fragments were filled using biotinylated nucleotides and ligated using T4 DNA ligase (NEB). After reversal of crosslinks, ligated DNA was purified and sheared to a length of ~300-500 bp using the Covaris ultrasonicator S220 (settings: 10% duty factor, 200 cycles per burst and a peak incident power of 140). Ligated fragments were pulled down using streptavidin beads (Invitrogen) and prepped for Illumina sequencing by subsequent end-repair, addition of A-overhangs and adapter ligation. Libraries were amplified for a total of 12 PCR cycles (45 sec at 98C, 12 cycles of 15 sec at 98C, 30 sec at 55C, 30 sec at 62C and a final extension of 5 min at 62C) and sequenced with the NOVASeq platform (Illumina), generating 100 bp paired-end sequence reads at the UCSD core facility.