GSM4681456: 3D7-GFPAP214-HP1glmS-ON-36-44-GFP-ChIP; Plasmodium falciparum 3D7; ChIP-Seq
Synchronised parasites were harvested at peak PfAP2-HC expression at 36-44 hpi at 4-5% parasitemia. Parasites were crosslinked with 1% formaldehyde for 10 min at 37 °C before quenching with 0.125 M glycine. The RBC membrane was lysed with 0.15% saponin and cytoplasmic lysis buffer (CLB: 20 mM Hepes, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.65% NP-40, 1 mM DTT, 1x protease inhibitor (Roche) was added to the parasite pellet to isolate nuclei. These were then snap-frozen in liquid nitrogen in CLB supplemented with 50% glycerol and stored at - 80 °C. Chromatin isolation, shearing and immunoprecipitation was performed according to previously published protocols (Filarsky et al. 2018). To prepare chromatin, frozen nuclei were thawed, pelleted and resuspended in 150 µl sonication buffer (50 mM Tris pH 8, 1% SDS, 10 mM EDTA, 1x protease inhibitor (Roche)), and were sonicated for 20 cycles of 30 sec ON/30 sec OFF (setting high, BioruptorTM Next Gen, Diagenode) to shear DNA to fragments of 100-600 bps. Fragment size was confirmed by de-crosslinking a 15 µl aliquot and visualising the purified DNA on a 2% agarose gel. ChIPs were performed by combining sonicated chromatin (500 ng DNA content) with either 1 µg mouse mAb α-GFP (Roche Diagnostics #11814460001) or 1 µg rabbit α-PfHP1 (Brancucci et al. 2014a). Reactions contained incubation buffer (5% Triton-X-100, 750 mM NaCl, 5 mM EDTA, 2.5 mM EGTA, 100 mM Hepes pH 7.4), 0.2% bovine serum albumin, 1x protease inhibitor (Roche) and 10 μl protA and 10 μl protG Dynabeads (Life Technologies, #10008D and #10009D, respectively). ChIP reactions were incubated overnight at 4 °C with rotation. Beads were washed for 5 min at 4 °C, with rotation, with 400 µl wash buffers as follows: 2x wash buffer 1 (0.1% SDS, 0.1% DOC, 1% Triton-X100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM Hepes pH 7.4), 1x wash buffer 2 (0.1% SDS, 0.1% DOC, 1% Triton-X100, 500 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM Hepes pH 7.4), 1x wash buffer 3 (250 mM LiCl, 0.5% DOC, 0.5% NP-40, 1 mM EDTA, 0.5 mM EGTA, 20 mM Hepes pH 7.4), 2x wash buffer 4 (1 mM EDTA, 0.5 mM EGTA, 20 mM Hepes pH 7.4). Immunoprecipitated chromatin was eluted from the beads by shaking at room temperature for 20 min in 200 µl elution buffer (1% SDS, 0.1 M NaHCO3) and de-crosslinked at 45 °C overnight in 1% SDS, 0.1 M NaHCO3 and 1 M NaCl. Simultaneously, 30 µl of sonicated input chromatin was de-crosslinked under the same conditions. DNA was purified with QIAquick MinElute PCR columns (Qiagen). Twenty separate α-GFP ChIPs or four separate α-PfHP1 ChIPs were combined, with the exception of 3D7/GFP-PfAP2-HC/PfHP1-mScarlet-glmS α-PfHP1 ChIPs for which eight separate reactions were combined. The obtained ChIPed DNA fragments were used to generate Illumina sequencing libraries according to Filarsky et al. (2018). In brief, 1 ng of α-PfHP1 ChIP, α-GFP ChIP, or input DNA were end-repaired with T4 DNA polymerase (NEB, M0203L), Klenow DNA polymerase (NEB, M0210L), and T4 Polynucleotide Kinase (NEB, M0201L). The 3'ends of end-repaired DNA was extended with an A-overhang with 3' to 5' exonuclease-deficient Klenow DNA polymerase (NEB, M0212L). The resulting fragments were ligated to Nextflex 6bp adaptors (Bio Scientific, #514122) with the use of T4 DNA ligase (Promega, M1804). The libraries were amplified using an AT-rich optimized KAPA protocol using KAPA HiFi HotStart ready mix (KAPA Biosystems, KM2602), NextFlex primer mix (Bio Scientific, #514122) with the following PCR program: 98°C for 2 min; four cycles of 98°C for 20 sec, 62°C for 3 min; 62°C for 5 min. The fragments originating from mono-nucleosomes + 125 bp NextFlex adapter were selected using 2% E-Gel Size Select agarose gels (Invitrogen, #G6610-02) and amplified by PCR for nine cycles using the above conditions. Libraries were purified and adapter dimers removed with Agencourt AMPure XP beads purification using a 1:1 library:beads ratio (Beckman Coulter, #A63880). ChIP-seq libraries were sequenced on the Illumina NextSeq 500 system with a 20% phiX spike-in (Illumina, FC-110-3001) to generate 75 bp single-end reads (NextSeq 500/550 High Output v2 kit).
GEO Accession
GSM4681456