GSM5174222: HiC_3D7_PfSETvs-KO_10-16h_rep2; Plasmodium falciparum 3D7; Hi-C
For Hi-C study, parasites were crosslinked by 1% formaldehyde for 10 min at room temperature followed by addition of 0.125 M glycine to stop crosslink. In situ Hi-C was performed as previously described with moderate modifications. Briefly, fixed parasites were lysed in 5 ml ice-cold Hi-C lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2% NP-40, 1X protease inhibitors) and rotated at 4 ℃ for 1 h. Pelleted nuclei were resuspended with 300 μl H2O, 44 μl of 10×NEBuffer 2 and 38 μl of 1% SDS and incubated at 65 °C for 10 minutes. 44 μL of 10% Triton X-100 were added and samples were rotated at 37 ℃ for 60 minutes to quench the SDS. To fragment DNA, 350 U endonucleases DpnII (NEB, R0543M) was added to the samples and samples were incubated at 37 ℃ overnight. Digested DNA was filled in the restriction fragment overhangs with Klenow (NEB, M0210L) and the DNA ends was marked with biotin. Chromatin was then ligated with T4 DNA ligase (thermo, EL0013) for 4 h at 16  ℃. DNAs were sonicated to reduce their size to 300-500 bp and immobilized on Dynabeads M-280 Streptavidin (Invitrogen,11205D).The process of ends repair, A-tailing and adaptor ligation referred to ChIP-seq method, the Hi-C library was amplified for 12-13 cycles and the PCR product was loaded on 1% agarose and size selected the 300-600bp band for sequencing. Then, Hi-C libraries were sequenced to generate at least 45 million paired-end reads (150 bp in length) per each donor/condition in a Novaseq 6000 platform (Illumina).