Name: GSM6106375
Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Parasites were crosslinked at a final concentration of 1% PFA for 10 min at 37°C and subsequently quenched with 125 mM glycine for 5 min on ice. The cells were then spun down and lysed using 0.075% saponin in PBS. After lysis the cells were washed twice with PBS and then resuspended in cold lysisbuffer (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA pH 8.0, 0.1 mM EGTA pH 8.0, 1 mM DTT, 1x Protease Inhibitor (Roche); 1ml per 5*10^7 cells). The mix was then transferred to a tissue grinder and incubated on ice for 30 min. Then Igepal was added to a final concentration of 0.25% and cells were lysed with 100 strokes. The resulting free nuclei were again pelleted and lysed by SDS lysis buffer (1 % SDS, 10 mM EDTA, 50 mM Tris pH 8.1, 1x Protease Inhibitor). The DNA was then sheared by sonication (2 times 8 min with 30s ON 30s OFF cycles). The sheared material was spun and the supernatant was transferred and diluted 1:10 with ChIP dilution buffer (0.01 % SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 150 mM NaCl). The diluted chromatin was then precleared by BSA-blocked agarose G beads (GE Healthcare) for 1h. Subsequently 50µl material were set aside as input. For the IP 15µl agarose G beads were used per antibody per sample. The IP was incubated under agitation at 4°C over night and thereafter washed sequentially with different buffers: Low Salt Wash Buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), then High Salt Wash Buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), then LiCl Wash Buffer (0.25 M LiCl, 1 % Igepal, 1 % Sodium-Desoxycholat, 1 mM EDTA, 10 mM Tris-HCl pH 8.1), then twice at RT with TE (10 mM Tris-HCl pH 8, 1 mM EDTA pH 8). The immunoprecipitated chromatin was eluted from the beads with 100 µl Elution Buffer (2 % SDS, 200 mM NaHCO3) for 15 min at RT, twice. The Input sample was also diluted to the same volume with Elution Buffer. 500 mM NaCl were added for decrosslinking (45 °C over night). Finally, the proteins were digested by addition of 2 µl Proteinase K (Thermo Fisher Scientific) for 1 h at 37 °C, and DNA was purified using the MinElute Kit (QIAGEN 28006). Libraries were generated using the Accel-NGS (TM) 2S Plus DNA Library Kit for Illumina platforms (SWI Swift Bioscience) and Accel-NGS (TM) 2S Indexing Kit (SWI Swift Bioscience) according to the manufacturer's guidelines. AmpureXP beads (Beckman Coulter) were used for the purification steps and the amplification step was performed with 15 cycles using the KAPA Hifi PCR kit (KK2101; Roche) according to published procedures (PMID: 26067602). directional paired-end ChIP-Seq