GSM7727908: nos-Gal4 driven control KD input; Drosophila melanogaster; ChIP-Seq
Ovaries were crosslinked with 1% formaldehyde in PBS for 10 min, then quenched with Glycine (final concentration 25 mM). Frozen ovaries were dounced in RIPA buffer and then sonicated (Bioruptor sonicator) to a desired fragment sizes of 200–800 bp. The supernatants were first precleared for 2 h at 4°C using Protein G Dynabeads (Invitrogen). Precleared samples were immunoprecipitated with anti-H3K9me3 (abcam, ab8898) antibodies for 3-5 h at 4°C, then 50 μl Protein G Dynabeads were added, and samples were further incubated overnight at 4°C. Beads were washed 3x 10 minutes in LiCL buffer, followed by proteinase K treatment for 2 h at 55°C and then overnight at 65°C. DNA was extracted by standard phenol-chloroform extraction. ChIP-seq libraries were prepared using NEBNext Ultra DNA Library Prep Kit Illumina (NEB), PE50