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Sample GSM4138018 Query DataSets for GSM4138018
Status Public on Mar 17, 2020
Title Input_S2 line_FLAG-Usp_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-Usp expression under Act5C promoter
isoform of nuclear receptor: D. melanogaster Usp full length (1-508 aa)
treatment: 0.3 uM 20-hydroxyecdysone (20E) for 1 hour
chip antibody: input chromatin (before precipitation)
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
 
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.
 
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 SAMN13107762
SRA SRX7050430

Supplementary file Size Download File type/resource
GSM4138018_Input_S2_line_FLAG-Usp_20E-1h.bigwig 13.7 Mb (ftp)(http) BIGWIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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