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| Status |
Public on Mar 17, 2020 |
| Title |
ChIP-FLAG_S2 line_FLAG-BioID2-EcR_20E-1h |
| Sample type |
SRA |
| |
|
| Source name |
S2 Schneider cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line/tissue: S2 Schneider cells
expression of nuclear receptor: 3xFLAG-BioID2-EcR expression under Act5C promoter
isoform of nuclear receptor: D. melanogaster EcR-A isoform full length (1-849 aa) fused with BioID2 through a 25nm GS linker
treatment: 0.3 uM 20-hydroxyecdysone (20E) for 1 hour
chip antibody: Monoclonal ANTI-FLAG® M2 antibody produced in mouse F1804 (Sigma-Aldrich)
|
| Treatment protocol |
Cells were co-transfected by expression contructs and pCoBlast plasmid gaving blasticidin resistance in 1 to 20 molar ratio using Effectene transfection reagent (Qiagen). Stable cell lines were selected and cultivated in blasticidin-containing S2 medium (20 ug/ml final concentration).
|
| Growth protocol |
Drosophila Schneider line 2 (S2) cells were maintained at 25°C in the Schneider’s insect medium (Sigma) containing 10% FBS (HyClone).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Crosslinking was performed with 1% formaldehyde for 10 min at RT and stopped by adding glycine. After washings, chromatin was lysed in SDS-containing buffer (50 mM HEPESKOH, pH 7.9; 140 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na deoxycholate, 0.1% SDS) with protease inhibitors cocktail (Roche) and sheared to 500-bp fragments by sonication. Sonicated chromatin was centrifuged twice at 16 000 g for 20 min, and used in immunoprecipitation experiments. For one experiment, about 5 ug of antibodies and 8 uL of MabSelect sepharose (GE healthcare) were taken; BSA were added to a final concentration of 1%. The precipitated chromatin was sequentially washed twice with SDS-containing buffer and with TE buffer (20mM Tris-HCl, pH 8.0; 1mM EDTA). Precipitated chromatin complexes were eluted by sequential incubation in two portions of elution buffer (50mM Tris-HCl, pH 8.0; 1mM EDTA, 1% SDS), 30 min each, at room temperature. The eluted chromatin solution was supplemented with 5 M NaCl (16 uL per 500 uL sample) and incubated at 65°C for 16 h on a thermoshaker for de-crosslinking. De-crosslinked chromatin was subjected to proteinase treatment (3 uL of proteinase K and 5 uL of 0.5M EDTA per 500 uL sample) and incubated at 55°C for 4 h on thermoshaker. DNA was extracted with a phenol/chloroform mixture and precipitated with isopropanol. The precipitate was dissolved in TE buffer and subjected to library preparation.
DNA libraries were prepared for sequencing using standard Illumina protocols
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| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
bcl2fastq v2.17.1.14 Conversion Software (Illumina)
ChIP-seq reads were aligned to the dm6 genome by bowtie2 v2.3.4.2 (using UseGalaxy.eu online tools)
Genome_build: dm6 (Drosophila melanogaster Release 6 plus ISO1 MT)
Supplementary_files_format_and_content: BigWig files were generated using bamCoverage 3.0.2. Scores represent number of reads normalized by the size of the library.
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| |
|
| Submission date |
Oct 24, 2019 |
| Last update date |
Mar 20, 2020 |
| Contact name |
Nadezhda E Vorobyeva |
| E-mail(s) |
nvorobyova@gmail.com
|
| Phone |
+79262790219
|
| Organization name |
Institute of Gene Biology RAS
|
| Street address |
Vavilova 34/5
|
| City |
Moscow |
| ZIP/Postal code |
119334 |
| Country |
Russia |
| |
|
| Platform ID |
GPL25244 |
| Series (1) |
| GSE139316 |
Employing proximity-dependent ligation techniques to estimate EcR/Usp molecular partners in Drosophila |
|
| Relations |
| BioSample |
SAMN13107775 |
| SRA |
SRX7050417 |
