GSM7144058: 3D7, iKO, induction24, 69hpi, Rep2, Mnase; Plasmodium falciparum; MNase-Seq
Parasites were crosslinked for 15 min in 1 % formaldehyde and stopped with 0.125M glycine. Erythrocytes were lysed with 0.1 % saponin and nuclei were isolated as in Josling et al. 2015. Nuclei were resuspended in 75 µl 50 mM Tris pH7.4/4 mM MgCl2/1 mM CaCl2/0.075 % NP40/1 mM DTT, PIC with 0.75 U Micrococcal nuclease (Worthington Biochemicals Corporation) and 50 U Exonuclease III (NEB). Each sample was aliquoted in 3 x 25 µl and incubated for 2.5 / 10 min at 37 °C under agitation (low/high digestion), before the reaction was stopped by adding 25 µl 2 % Triton X100/0.6 % SDS/300 mM NaCl/6 mM EDTA/PIC. and placed at 4 °C. Decrosslinking was performed at 45 °C overnight after adjusting to 1 % SDS/0.1 M NaHCO3/0.5 M NaCl. Proteins were digested by addition of 40 µg of Proteinase K (Zymo Research) and incubation for 1 h at 55 °C. Subsequently, DNA was isolated using EXTRACTME DNA clean-up kit and Micro Spin columns (Blirt) as manufacturer recommends. For low digested samples, DNA fragments >700 bp were removed using SPRI beads, no size-selection step was applied for high digested samples. 1 ng of high/low digested samples was used as input for NEBNext Ultra II DNA library prep kit (NEB), according to manufacturer's protocol.