GSM8724590: mod[07570]_H3K9me3 ChIP DNA; Drosophila melanogaster; ChIP-Seq
Approximately 50 μl ovaries were crosslinked with 1% formaldehyde in PBS for 10 min at room temperature, quenched with 150 mM glycine for 5 min, and washed three times, 5 min each, in PBT. Lysis was performed in 500 µl of lysis buffer (10 mM Tris-HCl at pH 8, 100 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 0.5% N lauroylsarcosine, 1 mM DTT and 1x Halt Protease Inhibitor from Thermo). Ovaries were homogenized on a 1 ml Wheaton homogenizer with 5 loose and 5 tight strokes and kept on ice for 30 min. Lysates were prepared as 100 μl aliquots and sonicated on a Bioruptor (Diagenode) for 10 cycles at high strength with 30 s ON and 45 s OFF pulses. The lysates were then pooled, and the volume was adjusted to 1 ml with lysis buffer, followed by centrifugation at 12,000 rpm for 10 min at 4°C. The supernatants containing the soluble chromatin were transferred to new tubes, and 100 μl was saved as input control. Immunoprecipitation was carried out by adding 2 μg of anti-H3K9me3 (Abcam, ab8898) to the respective supernatants and incubating overnight on a rotating wheel at 4°C. Protein A/G Magnetic Beads (ThermoFisher, #88802) were added to the supernatant-antibody mix, and the samples were incubated for an additional 3 hr at 4°C. The ChIP samples were washed five times: once with low-salt buffer (20 mM Tris-HCl at pH 8, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, and 0.1% Triton-X 100), twice with high-salt buffer (20 mM Tris-HCl at pH 8, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, and 0.1% Triton-X 100), once with LiCl buffer (10 mM Tris-HCl at pH 8, 1 mM EDTA, 1% NP40, 1% deoxycholate and 250 mM LiCl) and once with TE buffer (10 mM Tris-HCl at pH 8 and 1 mM EDTA). Then, the beads were resuspended in 100 μl of elution buffer (50 mM Tris-HCl at pH 8, 10 mM EDTA, 1% SDS, and 300 mM NaCl). The input samples were thawed, mixed with an equal volume of elution buffer, and processed in parallel with the ChIP samples. Crosslinking was reversed by incubating the beads and inputs overnight at 65°C. The samples were sequentially treated with 2 μl of RNAseA+T1 (Thermo, EN0551) for 30 min at 37°C, and 5 μl of Proteinase K (Thermo, EO0491) for 2 hr at 37°C, and the immunoprecipitated DNA was then extracted using a PCR purification kit (Qiagen). ChIP was repeated thrice with three independent biological replicates. One replicate was sequenced using the Illumina SE150 strategy, and all three biological replicates were used for qPCR. Libraries were prepared by Novogene using the NEBNext Ultra II DNA Library Prep Kit for Illumina