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| Status |
Public on Jun 26, 2023 |
| Title |
sfGFP-GAF degrad, H3K9me3 IP, 1 |
| Sample type |
SRA |
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| Source name |
embryo
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: embryo
developmental stage: 2-2.5 hr AEL
genotype: nos-degradFP/His2Av-RFP (II); sfGFP-GAF(N) (III)
chip antibody: anti-H3K9me3 (Active motif #39161)
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| Growth protocol |
All stocks were grown on molasses food at 25ºC.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed as described previously (Blythe and Wieschaus 2015) on hand selected 2-2.5 hr AEL embryos from: sfGFP-GAF/+ and sfGFP-GAFSDPQ/+ females, nos-degradFP/His2Av-RFP (II); sfGFP-GAF (N) (III) and His2Av-RFP (II); sfGFP-GAF (N) (III) females, and nos-DBD-sfGFP/+ females. Briefly, 200-400 embryos were collected, dechorionated in 50% bleach for 3 min, fixed for 15 min in 4% formaldehyde and then lysed in 1 mL of RIPA buffer (50 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, and 150 mM NaCl). The fixed chromatin was then sonicated for 20 s 11 times at 20% output and full duty cycle (Branson Sonifier 250). Chromatin was incubated with 6 μg of anti-GFP antibody (Abcam #ab290) or 10 μl anti-H3K9me3 antibody (Active Motif, # 39162) overnight at 4°C , and then bound to 50 μl of Protein A magnetic beads (Dynabeads Protein A, Thermo Scientific). The purified chromatin was then washed, eluted, and treated with 90 μg of RNaseA (37°C, for 30 min) and 100 μg of Proteinase K (65°C, overnight). The DNA was purified using phenol/chloroform extraction and concentrated by ethanol precipitation. Each sample was resuspended in 25 μl of water.
Sequencing libraries were made using the NEB Next Ultra II library kit. For sfGFP-GAFSDPQ ChIP-seq, libraries were sequenced on the Illumina NextSeq 500 using 75bp single-end reads at the Northwestern Sequencing Core (NUCore). For DBD-sfGFP ChIP-seq, libraries were sequenced on the Illumina HiSeq 4000 using 50bp single-end reads at the Northwestern Sequencing Core (NUCore). For H3K9me3 ChIP-seq, libraries were sequenced on the Illumina NovaSeq 6000 using 150bp paired-end reads at the UW Madison Biotechnology Center.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
KV1
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| Data processing |
ChIP-seq data was aligned using bowtie 2 v2.3.5 without the following default parameters to retain multimapping reads: --no-mixed --no-discordant
Replicates were merged into a single bigWig file. bamCompare was used with the following parameters --scaleFactorsMethod SES, --operation log2, to generate bigWig files of the H3K9me3 IP signal normalized to the input
ChIP-seq peak calling was performed using MACS v2 with the following parameters: -g 1.2e8, --call-summits.
To focus analysis on robust, high-quality peaks, we used 100 bp up- and downstream of peak summits, and retained only peaks that were detected in both replicates and overlapped by at least 100 bp.
Assembly: dm6
Supplementary files format and content: Replicates were merged into a single bigWig file.
Supplementary files format and content: bed files containing peaks. Contains only peaks that were detected in both replicates.
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| Submission date |
Nov 15, 2022 |
| Last update date |
Jun 26, 2023 |
| Contact name |
Melissa M Harrison |
| E-mail(s) |
mharrison3@wisc.edu
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| Organization name |
University of Wisconsin Madison
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| Department |
Biomolecular Chemistry
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| Lab |
1135 Biochemical Sciences Bldg
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| Street address |
420 Henry Mall
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| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
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| Platform ID |
GPL25244 |
| Series (2) |
| GSE218019 |
Localization of the pioneer factor GAF to subnuclear foci is driven by DNA binding and required to silence satellite repeat expression [ChIP-Seq] |
| GSE218020 |
Localization of the Drosophila pioneer factor GAF to subnuclear foci is driven by DNA binding and required to silence satellite repeat expression |
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| Relations |
| BioSample |
SAMN31740970 |
| SRA |
SRX18273817 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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