Tethered Conformation Capture Hi-C of P. falciparum stage II/III gametocytes
P. falciparum cultures were crosslinked for 25 minutes at 37C in 1.25% formaldehyde in a total volume between 1 and 10 ml, depending on the number of parasites harvested. The reaction was quenched for 15 min at 37C, followed by extensive washes to remove cellular debris. To map the inter- and intrachromosomal contact counts, crosslinked parasites were subjected to the tethered conformation capture: proteins were first biotinylated and the DNA was digested using the restriction enzyme MboI. Fragments were captured on streptavidin-coated beads, ligated under dilute conditions, de-crosslinked and sheared to approximately 500 bp fragments. Libraries were generated using NEBNext Multiplex Oligos for Illumina and Kapa HiFi Hotstart ReadyMix (Kapa Biosystems) and amplified for a total of 12 PCR cycles (15s at 98ĄC, 30s at 55ĄC, 30s at 62ĄC) and were subsequently sequenced using the Illumina HiSeq2500 platform, generating 50 bp paired-end reads.