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| Status |
Public on Jun 26, 2023 |
| Title |
2-3H_PQ-mut_input_rep1 |
| Sample type |
SRA |
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| Source name |
embryo
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: embryo
genotype: sfGFP-GAFS-delta-PQ/sfGFP-GAFS-delta-PQ
developmental stage: 2-3 hour embryo
chip antibody: input
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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 2-3 hr AEL embryos from: sfGFP-GAF/+ and sfGFP-GAFSDPQ/sfGFP-GAFSDPQ 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) 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. Libraries were sequenced on the Illumina NextSeq 500 using 75bp single-end reads at the Northwestern Sequencing Core (NUCore).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
MG60
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| Data processing |
Bowtie2 version 2.4.4 was used to align ChIP-seq reads to the Drosophila melanogaster genome (version dm6) with the following non-default parameters: -k 2 --very-sensitive --no-mixed --no-discordant -X 5000.
Aligned reads were filtered to include only reads with a mapping quality score > 30. Reads mapping to unplaced scaffolds or the mitochondrial genome were discarded.
. Peak calling was performed using MACS2 version 2.2 with parameters -g dm --call-summits. Only peaks that were detected in all replicates were considered in downstream analysis.
bigWig files were generated using deepTools bamCoverage version 3.5.1 with parameters –binSize 10. bigWig files were z-score normalized by subtracting the genome-wide average of all bins from each bin value and dividing by the standard deviation of all bins. Z-score normalized signal was multiplied by a scaling factor derived from the percentage of reads aligning to the spike-in genome.
Assembly: dm6
Supplementary files format and content: Replicates were merged into a single bigWig file. BigWig files contain z-score normalized read depth that scaled according to the percentage of spike-in reads.
Supplementary files format and content: narrowPeak files containing only peaks that were detected in both replicates.
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| Submission date |
May 02, 2023 |
| 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 |
GPL19132 |
| 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 |
SAMN34542950 |
| SRA |
SRX20200948 |
| 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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