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| Status |
Public on Aug 22, 2021 |
| Title |
ChIP_SuHw_stock_SuHw_vf__ovary_1-13_chambers |
| Sample type |
SRA |
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| Source name |
Ovaries were dissected from females 32-34 hours after eclosion (containing egg chambers corresponding to 1-13 stages).
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: su(Hw)v/f stock (a gift of A. Golovnin)
Sex: female
tissue: ovary
chip antibodies: raised in a current study using antigen corresponding to N-part of Su(Hw) (antibodies raised with the same epitope was described in Golovnin et al (2008)EMBO Rep 9:440-445.
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| Treatment protocol |
Total DNA analysis, RNA-Seq and ChIP-Seqs by anti-Su(Hw) antibodies were performed using ovaries with egg chambers corresponding to 1-13 stages, which were dissected from Oregon-R-modENCODE and su(hw)v/f Drosophila stocks. FAIRE-Seq and ChIP-Seqs by anti-Su(Hw), anti-ORC2, anti-CDC6, anti-Brm, anti-ISWI, anti-CHD1, anti-Mi2, anti-Gcn5 antibodies were performed using ovaries with egg chambers corresponding to 1-8 stages dissected from Oregon-R-modENCODE and su(hw)v/e8 Drosophila stocks.
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| Growth protocol |
The flies of Oregon-R-modENCODE (oregon) (corresponds to Bloomington stock 25211), su(Hw)v/E8 and su(Hw)v/f stocks (a kind gift of A. Golovnin laboratory) were used. All flies were raised at 25°C on standard agar medium.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP 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. Total RNA was extracted with the TRI reagent (Ambion). PolyA comprising RNA fraction was isolated with the NEBNext UltraTM II Directional RNA Library Prep Kit. FAIRE-Seq protocol was perfomed exactly as previously described in Simon et al (2012) Nat protocols, 7:256-267.
DNA and RNA-Seq 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 |
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| Data processing |
bcl2fastq v2.17.1.14 Conversion Software (Illumina)
ChIP-seq and FAIRE-Seq reads were aligned to the dm6 genome by bowtie2 v2.3.4.2 (using UseGalaxy.eu online tools)
Total DNA reads were aligned to the dm6 genome by HISAT2 (using UseGalaxy.eu online tools)
RNA-Seq data were analyzed with TopHat2 (using UseGalaxy.eu online tools)
Genome_build: dm6 (Drosophila melanogaster Release 6 plus ISO1 MT)
Supplementary_files_format_and_content: Aligned reads were sorted to have MAPQ>5. 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 |
Mar 15, 2021 |
| Last update date |
Aug 22, 2021 |
| Contact name |
Nadezhda E Vorobyeva |
| E-mail(s) |
nvorobyova@gmail.com
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| Phone |
+79262790219
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| Organization name |
Institute of Gene Biology RAS
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| Street address |
Vavilova 34/5
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| City |
Moscow |
| ZIP/Postal code |
119334 |
| Country |
Russia |
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| Platform ID |
GPL25244 |
| Series (1) |
| GSE168894 |
Role of Su(Hw) in DNA amplification in Drosophila ovaries |
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| Relations |
| BioSample |
SAMN18310770 |
| SRA |
SRX10340448 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5172001_ChIP_SuHw_stock_SuHw_vf_ovary_1-13_chambers.bigwig |
66.8 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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