GSM2491670: GDVDD_10h_OFF_HP1; Plasmodium falciparum; ChIP-Seq
Synchronized 3D7/GDV1-GFP-DD schizonts on and off Shield1 at 5% parasitemia were cross-linked by incubation with a final concentration of 1% formaldehyde for 15 min at 37ºC. The cross-linking reaction was quenched by addition of 0.125M glycine (final concentration). Nuclei were isolated by releasing parasites from iRBCs using 0.05% saponin followed by lysis in CLB (20 mM Hepes, 10mM KCl, 1mM EDTA, 1mM EGTA, 0.65% NP-40, 1mM DTT supplemented with protease inhibitor). Nuclei were washed and snap-frozen in CLB supplemented with 50% glycerol. To obtain fragmented chromatin for ChIP, nuclei were resuspended in sonication buffer (50 mM Tris pH:8.0, 1% SDS, 10mM EDTA, supplemented with protease inhibitor) and sonicated for 20 cycles of 30 sec ON/30 sec OFF (setting high, BioruptorTM, Diagenode). Fragment sizes ranged from 200-600 bp as determined by decrosslinking a 50μl aliquot and running the purified DNA on a 1.5% agarose gel.    ChIPs were performed by incubating ~500 ng (DNA-content) sonicated chromatin from 3D7/GDV1-GFP-DD on and off Shield1 overnight while rotating at 4°C with 1 μg mouse anti-GFP (Roche Diagnostics, #11814460001) or 1 μg rabbit anti-PfHP1 (Brancucci et al. 2014), respectively, in incubation buffer (5% Triton-X-100, 750mM NaCl, 5mM EDTA, 2.5mM EGTA, 100mM Hepes pH 7.6) with 10 μl protA and 10 μl protG Dynabeads (Life Technologies, 10008D and 10009D). Beads were washed for 5 min at 4°C while rotating with 400 μl of the following wash buffers: 2x wash buffer 1 (0.1% SDS, 0.1% DOC, 1.0% Triton-X100, 1mM EDTA, 0.5mM EGTA, 20mM Hepes pH 7.6), 1x wash buffer 2 (0.1% SDS, 0.1% DOC, 1.0% Triton-X100, 500mM NaCl, 1mM EDTA, 0.5mM EGTA, 20 mM Hepes pH 7.6), 1x wash buffer 3 (250mM LiCl, 0.5% DOC, 0.5% NP-40, 1mM EDTA, 0.5mM EGTA, 20mM Hepes pH7.6), 2x wash buffer 4 (1mM EDTA, 0.5mM EGTA, 20 mM Hepes pH7.6). The immunoprecipitated chromatin was eluted in 200 μl elution buffer (1% SDS, 0.1M NaHCO3) while rotating for 20 min at RT and decrosslinked in decrosslinking buffer (1%SDS, 0.1M NaHCO3, 1M NaCl) in a 45°C shaking heat-block overnight. In parallel, 30 μl of sonicated input chromatin was decrosslinked under the same conditions. The DNA was purified over QIAquick MinElute PCR columns (Qiagen) and eight separate anti-GFP or two anti PfHP1ChIPs were pooled over one column. For each sequencing library generated from 3D7/GDV1-GFP-DD parasites 1ng (anti-mouse GFP antibody) , 5ng (anti-HP1 antibody) of ChIP DNA or 1 ng of the input DNA were end repaired, extended with 3′ A-overhangs, and ligated to barcoded NextFlex adapters (Bio Scientific,  #514122). Libraries were amplified using the KAPA HiFi HotStart ready mix (KAPA Biosystems) and NextFlex primer mix (Bio Scientific, #514122) as follows: 98 °C for 2 min; 4 cycles of 98 °C for 20 sec, 62 °C for 3 min; 62 °C for 5 min. Amplified libraries were gel size-selected for 225-325 bp (mono-nucleosomes + ~125 bp NextFlex adapter) using 2% E-Gel Size Select agarose gels (Invitrogen, #G6610-02) and again amplified as described above for 10 cycles. Libraries were purified and adapter dimers removed by Agencourt AMPure XP beads purification using a1:1 library beads ratio (Beckman Coulter, #A63880).