GSM2584446: M1BP RNAi Hi-C biological rep B technical rep 1; Drosophila melanogaster; Hi-C
S2 cells have been fixed with 2% formaldehyde for 10 minutes at room temperature. Glycine at 125 mM final concentration has been added to the plates followed by 5 minutes incubation. After two washes in PBS, cells have been scraped off the plate and pelleted. Each pellet (10-50 million of cells) has been resuspended in 1 ml of lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% IGEPAL CA-630) and nuclei have been extracted by sonication (Arrigoni et al. 2016) using Covaris E220 sonicator (settings: 75W peak power, 2% duty factor, 200 cycles/burst), for 60-90 seconds until about 70% of intact nuclei have been released. 10-50 ng of DNA bound to beads have been used for library preparation using a modification of the NEBNext Ultra II DNA library preparation workflow (NEB, E7645). DNA bound to beads have been end-repaired, A-tailed, adaptor-ligated using manufacturer’s instruction. Beads were reclaimed on a magnet, washed once in EB and eluted at 98 °C for 10 minutes. DNA has been USER-treated to open the adapters in meantime to PCR amplification.
GEO Accession
GSM2584446