|
| Status |
Public on Feb 22, 2018 |
| Title |
H3K27ac_IP_wing_replicate1 |
| Sample type |
SRA |
| |
|
| Source name |
Developmental Stage Imaginal Disc wing disc
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: Developmental Stage Imaginal Disc wing disc
developmental stage: Third instar larvae
antibody: H3K27ac
genotype: wildtype
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
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.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
To trim adaptor sequences, Cutadapt ver. 1.2.1 (Martin 2011) was used. For the alignment, the trimmed reads were mapped to the Drosophila genome (dm3 assembly) using Bowtie ver. 12.0 (Langmead et al. 2009) with a parameter (-m 1).
Consistency of replicates was checked by genome-wide correlation analysis and by visual inspection.
Only uniquely aligned reads were used for the entire analyses. To obtain input normalized profiles, callpeak function was used with parameters (-B --nomodel --extsize 75 --SPMR) and bgdcmp function (-m FE) using MACS. Ver.2 (Zhang et al. 2008).
Genome_build: dm3
Supplementary_files_format_and_content: tdf (wig) and IP fold enrichment profile compared to input
|
| |
|
| Submission date |
Nov 09, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Mitzi Kuroda |
| E-mail(s) |
mkuroda@genetics.med.harvard.edu
|
| Organization name |
Brigham & Women's Hospital
|
| Lab |
NRB168
|
| Street address |
77 Ave. Louis Pasteur
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL13304 |
| Series (1) |
| GSE104059 |
Bivalent complexes of PRC1 with orthologs of BRD4 and MOZ/MORF target developmental genes in Drosophila |
|
| Relations |
| BioSample |
SAMN08007099 |
| SRA |
SRX3380809 |
