title: GSM6436691: ChIP-seq_wt_PfTau95_S_input; Plasmodium falciparum; ChIP-Seq
library_construction_protocol: Parasite culture containing 1 × 10^9 ring-stage parasites(10-15hpi) was harvested and cross-linked immediately with 1% paraformaldehyde with rotation for 10 min at 37 ℃, and quenched with 0.125 M glycine for 5 min at 4 ℃. The fixed parasites were isolated from iRBCs with 0.15% saponin for 15 min on ice. After washed with cold 1 x PBS, the nuclei were isolated by incubation with 2 ml of cold Lysis Buffer (10 mM KCl, 10 mM HEPES, 0.1 mM EDTA, 0.1mM EGTA, 1mM DTT, 0.25% N-P40, 1 x protease inhibitors cocktail) for 30 min on ice, and followed by dounce homogenization for 200 strokes. Then, the nuclei were resuspended in 200 l of SDS Lysis Buffer (1 % SDS, 10 mM EDTA, 50 mM Tris-HCl) for 20 minutes at 5 % duty factor, 200 cycles per burst, and 75 W of peak incident power with a Covaris M220 sonicator to generate 200-600 bp fragments. The protein-DNA complexes were then immunoprecipitated by using 0.05 μg of mouse anti-GFP antibody (Abcam). The complex of protein A/G agarose-antibody-chromatin was washed once under agitation with High Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, 500 mM NaCl), Low Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8.0, 150 mM NaCl) and LiCl Immune Complex Wash Buffer (0.25 M LiCl, 1% NP-40, 1% deoxycolate, 1 mM EDTA, 10 mM Tris–HCl pH 8.0) for 5 min at 4°C , then washed twice at room temperature with TE Buffer (1 mM EDTA, 10 mM Tris–HCl). Protein-DNA complex was reversed cross-linking with RNase A at 37°C for 30 min and Proteinase K at 45°C for 2 h. ChIP DNA were obtained using MinElute PCR purification kit (Qiagen, 28006). The DNA sequencing libraries were created with the following successive six steps: end repair (Invitrogen No.16123), dA tailing (NEB No.M0212L), adaptor ligation (NEB No.M2200L), size selection (Beckman, Agencourt AMPure XP), PCR amplification (KAPA Biosystems, KB2500) and sequenced on an Illumina HiSeq Xten systerm to generate 150 bp pair-end reads.