GSM7785603: FgPH-1, in vitro MS-sucrose, 3dpi, biol rep 1; Fusarium graminearum; Hi-C
Cultures were amended with 1.8 % formaldehyde final for 15 min prior harvest under constant agitation, and mycelia were collected by filtratiion before flash freezing in liquid nitrogen. Frozen fixed mycelium was ground for 4x45s at 30 Hz using a TissueLyzer (Qiagen) and placed on ice with 600 µL of HiC buffer (Tris-HCl pH8 15 mM, KCl 5 mM, NaCl 15 mM, MgCl2 3 mM, Igepal CA-630 0.2 %, Roche® cOmplete Mini EDTA-free protease inhibitor cocktail 1X) in 1.5 mL Diagenode microtubes. After eight cycles of gentle sonication (mode “low” – 15s ON/30s OFF) on a Bioruptor Standard Waterbath Sonicator (Diagenode), samples were filtered on Sefar Nitex (100 µm) by centrifugating 10 min at 4,700g and 4°C. Pellets were then carefully resuspended in one mL of nuclei buffer (Tris-HCl pH8 15 mM, sucrose 250 mM, KCl 5 mM, MgCl2 1 mM, glycerol 40 %, PMSF 1 mM, Roche® cOmplete Mini EDTA-free protease inhibitor cocktail 1X) and centrifuged at 7,000g for 5 min and 4°C. After a final wash with 0.5 mL of nuclei buffer, nuclei pellets were collected and stored at -80°C until in situ HiC sample preparation with the Arima-HiC technology, following the manufacturers protocol. Arima Library Preparation using Swift Biosciences® Accell-NGS® 2S Plus DNA Library Kit and the KAPA Library Amplification Kit with Primer Mix, following the manufacturer's instructions