GSM2849576: H3K27ac_IP_wing_replicate2; Drosophila melanogaster; ChIP-Seq
We followed the BioTAP-XL protocol as described in (Alekseyenko et al. 2015). In brief, 12-24 hr old embryos from transgenic fly lines expressing BioTAP-N-Br140 were collected and stored for up to 3 days at 4°C. Embryos were cross-linked with 3% formaldehyde, extracts were prepared as described, and snap-frozen in liquid nitrogen for storage at -80°C. 20 grams of embryos were used for each experiment. S2 cell lines stably expressing BioTAP-N-Br140, BioTAP-N-dBRD4, and dBRD4-C-BioTAP were grown at 26.5°C to a density of ∼1 × 107 cells per milliliter using 500 mL of HyClone CCM3 serum-free medium in 2.8-L Fernbach glass flasks with shaking at 90 rpm. Cross-linked nuclear extracts from S2 cells were prepared from a total of 5 × 109 cells. After sonication, tandem affinity purification was performed to purify the BioTAP-tagged bait along with its protein interaction partners and associated genomic DNA. Genomic localization was determined by high-throughput sequencing of libraries generated from the tandem affinity-purified material using the NEBNext ChIP-seq library preparation master mix set for Illumina (New England Biolabs, catalog no. E6240S) and TruSeq adaptors (Illumina). Imaginal disc ChIP-seq was performed as described in Langlais et al (Langlais et al. 2012) with some modifications. 50~100 wing discs or haltere and 3rd leg discs were collected from dissection of 3rd instar larvae, crosslinked in 2% formaldehyde and sonicated using a BioRuptor (Diagenode) for 15 cycles (30 sec on/30 sec off) on high power. 5% of the total volume of sonicated chromatin was saved for input. ChIP was performed with 1:200 dilutions of anti-H3K27me3 (Cell Signaling), anti-H3K27ac (Active Motif). DNA was purified from immunoprecipitated samples. Illumina ChIP-seq libraries for H3K27me3 and H3K27ac were prepared using the library preparation kit (New England Biolabs) and sequenced as described above. Libraries for sequencing were prepared using standard Illumina protocols.