GSM5070348: Input of NF54; Plasmodium falciparum; ChIP-Seq
Chromatin was obtained from saponin-extracted parasites at the late trophozoite or schizont stage, crosslinked with formaldehyde and lysed following the MAGnifyTM Chromatin Immunoprecipitation System (Life Technologies). Samples were sonicated using an M220 sonicator (Covaris) at 10% duty factor, 200 cycles per burst, 140 W of peak incident power for 10 min. Immunoprecipitations were performed overnight at 4ºC with 4 µg of chromatin and 8 µg of antibodies against H3K9me3 (Diagenode pAb-193–050) coupled to protein A/G magnetic beads provided in the kit. Washing, de-crosslinking and elution were performed following the MAGnifyTM Chromatin Immunoprecipitation System recommendations, but avoiding high temperatures that may result in denaturation of extremely AT-rich intergenic regions: de-crosslinking, proteinase K treatment and elution were performed at 45ºC (for 2 h, over-night, and 1.5 h, respectively). Libraries for Illumina sequencing were prepared from 5 ng of immunoprecipitated DNA using a protocol adapted to a genome with an extremely high AT-richness (Kensche et al., 2016. PMID: 26578577). In brief, after end-repair and addition of 3′ A-overhangs, NEBNext Multiplex Oligos for Illumina (NEB, E7335, E7500) were ligated. Purification steps were performed with Agencourt AMPure XP beads (Beckman Coulter, A63880). Libraries were amplified using the KAPA HiFi PCR Kit (KAPA Biosystems, KK2103) in KAPA HiFi Fidelity Buffer (5X) with the following conditions: 95°C for 3 min; 9 cycles at 98°C for 20 s and 62°C for 2 min 30 s; and 62 °C for 5 min. Amplified libraries were purified using 0.9X AMPure XP beads to remove adapter dimers. The library size was analyzed in a 4200 TapeStation System (Agilent Technologies).