Name: GSM7192233
Instrument: Illumina NovaSeq 6000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: The ChIP assay was performed as described (Pandey et al., 2020) with a few modifications. Parasite culture (50 ml) containing synchronized late-stage schizonts (~ 40 h.p.i.) at ~5% parasitemia was centrifuged at 900 g for 4 min and the cells washed once with PBS. To the cell pellet, 25 ml of 0.15% saponin in PBS was added and incubated on ice for 10 min followed by washing twice with cold PBS. Parasites were crosslinked for 10 min by adding methanol-free formaldehyde at 1% final concentration and incubated for 10 min at 37 °C with occasional shaking. The crosslinking reaction was quenched by adding 1.25 M glycine to achieve a final concentration of 0.125 M and incubated at 37 °C for another 5 min. Parasites were centrifuged for 10 min at 3250 g at 4 °C, washed three times with DPBS, snap frozen in liquid nitrogen and stored at -80 °C until further use. Frozen formaldehyde fixed parasites were thawed on ice for chromatin immunoprecipitation. One ml of nuclear extraction buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1mM DTT, 1x EDTA-free protease inhibitor cocktail [Roche]) was added to the tubes containing thawed parasites and incubated on ice for 30 min. After the incubation, 10% NP-40 was added to reach a final concentration of 0.25% and the parasites were lysed by passing through a 261/2 G needle seven times. Parasite nuclei were collected by centrifuging at 2500g for 10 min at 4 °C. Shearing of chromatin was carried out using the Covaris Ultra Sonicator (E220) for 14 min with the following settings; 5% duty cycle, 140 intensity peak incident power, 200 cycle per burst to obtain fragment size of 200 to 600 bp. Insoluble materials were removed by centrifuging the sheared chromatin for 10 min at 13500 g at 4 °C. 30 µl of fragmented chromatin were stored as input at -80°C. Fragmented chromatin was diluted 1:1 in ChIP dilution buffer (30 mM Tris-HCl pH 8.0, 0.1% SDS, 3 mM EDTA, 300 mM NaCl, 1% Triton X-100, EDTA-free protease inhibitor cocktail). Chromatin was incubated overnight with 6 µg of mentioned antibodies. The antibody-protein complex was recovered with protein A coupled to magnetic beads (Dynabeads, Invitrogen, Cat No. 10002D), followed by extensive washes with low salt immune complex wash buffer, high salt immune complex wash buffer (washes done at 4 °C) and TE buffer (washes done at RT). Chromatin was eluted with elution buffer (1% SDS, 0.1 M NaHCO3) at 45 °C for 30 min with shaking. Immunoprecipitated chromatin and input were reverse crosslinked overnight at 45 °C by adding 5 M NaCl to a final concentration of 0.5 M. Samples were treated with RNaseA for 30 min at 37 °C followed by a 2 h incubation at 45 °C with proteinase K (final concentration 66 µg/ml). DNA was purified using ChIP DNA clean & concentrator (Zymo Research, Cat. No. D5205). Libraries were prepared using NEBNext Ultra II DNA library kit following the manufacturer's instructions until the step of adapter ligation (Adapters were diluted at 1: 20 ratio). Adapter ligated libraries were purified using AmpureXP beads. The libraries were amplified for a total of 6 PCR cycles (2 min at 98 °C initial denaturation; 6 cycles of 30 s at 98 °C, 50 s at 62 °C, final extension 5 min at 62 °C) using the KAPA HiFi HotStart Ready Mix (Kapa Biosystems). Amplified libraries were purified and size selected for 350 bp inserts using AmpureXP beads and sequenced on the Illumina HiSeqX platform with 150 bp paired-end read layouts.