GSM5456739: Pcy_M_bloodstage_H3K914ac_ChIP; Plasmodium cynomolgi; ChIP-Seq
Native ChIP was carried out as described earlier {Hoeijmakers, 2012 }. In short: After saponin lysis of the red blood cells and/or lysis of the parasites, nuclei were separated using a 0.25 M sucrose buffer cushion. Native chromatin was prepared by MNase digestion and subsequent extraction with salt-free buffers (10 mM Tris pH 7.4, 1 mM EDTA; 1 mM Tris pH 7.4, 0.2 mM EDTA). Chromatin was diluted in 2×ChIP incubation buffer (100 mM NaCl, 20 mM Tris pH 7.4, 6 mM EDTA, 1% Triton X-100, 0.1% SDS). ∼400 ng DNA-containing chromatin was incubated with 1 µg antibody (H3K9/14ac (Diagenode, C15410005); H3K9me2 (Sigma-Aldrich, 07-441)) overnight at 4°C followed by the addition of 10 µl A/G beads (SantaCruz Biotechnology) and further incubation for 2 h. After washing with buffers containing 100, 150 and 250 mM NaCl, immuno-precipitated DNA was eluted and purified using PCR purification columns (Qiagen). 1-10 ng of ChIP-ed or input DNA were used to generate sequencing libraries. All samples were first end repaired, a 3' A-overhang added and NextFlex 6bp barcoded adapters (Bio Scientific) were ligated as described in. Adapter ligation was followed by 2 successive Agencourt AMPure XP bead purifications (Beckman Coulter) and library amplification by an in-house Plasmodium-optimized KAPA PCR amplication protocol. First, 2x KAPA HiFi HotStart ready-mix (KAPA Biosystems) and NextFlex primers were used for 4 cycles of PCR amplification (98 °C for 2 min; 4 cycles of 98 °C for 20 sec, 62 °C for 3 min; 62 °C for 5 min). Subsequently, amplified libraries were size selected for ~270 bp (mono-nucleosomes + ~125 bp 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. ChIP-seq and input 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
GSM5456739