title: GSM2127887: Pf_DCJ_H33Ty1_T10_H3core_xLCHIP; Plasmodium falciparum; ChIP-Seq
library_construction_protocol: xl anti-histone H3 ChIP (H3.3 and H3) protocol to extract and prepare the material:  Parasite cultures used for cross-linked ChIP were immediately treated with 1% formaldehyde (final concentration) and incubated at 37°C for 10 min while shaking. Crosslinking reaction was quenched by adding 0.125M glycine (final concentration). After erythrocyte and parasite lysis using 0.05% saponin and a hypotonic buffer (10 mM Tris pH 8.0, 3 mM MgCl2 , 0.2%  NP-40, Roche Protease Inhibitor Cocktail) respectively, formaldehyde-cross-linked nuclei were separated from cell debris using a 0.25 M sucrose buffer cushion as described in (Bártfai et al., 2010). Subsequent, crosslinked chromatin was prepared as described in (Kensche et al., 2015) with a few adjustments. In short, for generation of genome-wide PfH3.3 profiles, enzymatic MNase digestion of cross-linked chromatin was performed using digestion buffer (50mM Tris pH7.4, 4mM MgCl2, 1mM CaCl2, 0.075% NP40, Roche Protease Inhibitor Cocktail) with 0.5 U MNase (Worthington Biochemicals Corporation) in 150μl aliquots at 37 ºC. Optimal digestion times were empirically determined for each stage by test-digestion of a nuclear aliquot for varying times, to obtain primarily mono-nucleosomal DNA fragments. Digestion reactions were stopped by adding 150μl quenching solution (2% Triton X100, 0.6% SDS, 300 mM NaCl, 6 mM EDTA, Roche Protease Inhibitor Cocktail) and placing the samples on ice. Subsequently, digested chromatin was sonicated for 6 x 10 sec (setting low, BioruptorTM Next Gen, Diagenode) to free cross-linked chromatin from nuclear membranes. Approximately 200 ng of digested DNA-containing cross-linked chromatin was incubated in ChIP buffer (150mM NaCl, 20mM Tris pH 8.0, 2mM EDTA, 1% Triton X-100, 0.1% SDS, Roche Protease Inhibitor Cocktail ) with the respective antibody overnight at 4°C while rotating, followed by the addition of 10µl ProtA/G beads (Life Technologies, 10008D and 10009D) and further incubation at 4°C for 2 h. For the anti-histone H3 antibody (capturing histone PfH3 and PfH3.3) one ChIP reaction was performed per life cycle stage (1 µg antibody/ChIP).After washing with three different buffers (buffer1, 1x: 20mM Tris pH 8.0, 2mM EDTA, 1% Triton-X100, 0.1% SDS, 150mM NaCl; buffer 2, 2x: 20mM Tris pH 8.0, 2mM EDTA, 1% Triton-X100, 0.1% SDS, 500mM NaCl and buffer 3, 2x: 10mM Tris pH 8.0, 1mM EDTA) immunoprecipitated chromatin was eluted using 1% SDS and 0.1M NaHCO3. Immunoprecipitated chromatin was de-cross-linked (1% SDS, 0.1M NaHCO3, 1M NaCl) overnight at 45 °C shaking, followed by DNA isolation via QIAquick column purification (Qiagen). Sequencing libraries were prepared according to a Plasmodium-optimized library preparation procedure including KAPA polymerase-mediated PCR amplification For each sequencing library generated from endogenously PfH3.3-Ty1 expressing DCJ P. falciparum parasites 3 ng of ChIP DNA were end repaired, extended with 3’  A-overhangs, and ligated to barcoded NextFlex adapters (Bio Scientific) (Hoeijmakers et al., 2011). Subsequent library amplification was performed under similar conditions as described previously (Kensche et al., 2015) with a few adjustments: First, 2x KAPA HiFi HotStart ready-mix (KAPA Biosystems) and NextFlex primers were used for 4 cycles of PCR amplification (98°C for 2min; 4 cycles of 98°C for 20sec, 62°C for 3min; 62°C for 5 min). Subsequently, amplified libraries were size selected for 270 bp (mono-nucleosomes + ~125bp NextFlex adapter) using 2% E-Gel Size Select Agarose Gels (Thermo Fisher Scientific) and again amplified as described above for 9 cycles. To deplete adapter dimers and clean-up the DNA, libraries were subsequently purified with Agencourt AMPure XP bead (Beckman Coulter) (1:1 library beads ratio) before sequencing.
attributes.tag: GEO Accession
attributes.value: GSM2127887