GSM6732888: sfGFP-GAF control, H3K9me3 Input, 2; Drosophila melanogaster; ChIP-Seq
ChIP was performed as described previously (Blythe and Wieschaus 2015) on hand selected 2-2.5 hr AEL embryos from: sfGFP-GAF/+ and sfGFP-GAFSDPQ/+ females, nos-degradFP/His2Av-RFP (II); sfGFP-GAF (N) (III) and His2Av-RFP (II); sfGFP-GAF (N) (III) females, and nos-DBD-sfGFP/+ females. Briefly, 200-400 embryos were collected, dechorionated in 50% bleach for 3 min, fixed for 15 min in 4% formaldehyde and then lysed in 1 mL of RIPA buffer (50 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, and 150 mM NaCl). The fixed chromatin was then sonicated for 20 s 11 times at 20% output and full duty cycle (Branson Sonifier 250). Chromatin was incubated with 6 μg of anti-GFP antibody (Abcam #ab290) or 10 μl anti-H3K9me3 antibody (Active Motif, # 39162) overnight at 4°C , and then bound to 50 μl of Protein A magnetic beads (Dynabeads Protein A, Thermo Scientific). The purified chromatin was then washed, eluted, and treated with 90 μg of RNaseA (37°C, for 30 min) and 100 μg of Proteinase K (65°C, overnight). The DNA was purified using phenol/chloroform extraction and concentrated by ethanol precipitation. Each sample was resuspended in 25 μl of water. Sequencing libraries were made using the NEB Next Ultra II library kit. For sfGFP-GAFSDPQ ChIP-seq, libraries were sequenced on the Illumina NextSeq 500 using 75bp single-end reads at the Northwestern Sequencing Core (NUCore). For DBD-sfGFP ChIP-seq, libraries were sequenced on the Illumina HiSeq 4000 using 50bp single-end reads at the Northwestern Sequencing Core (NUCore). For H3K9me3 ChIP-seq, libraries were sequenced on the Illumina NovaSeq 6000 using 150bp paired-end reads at the UW Madison Biotechnology Center.