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| Status |
Public on May 02, 2023 |
| Title |
S2-WT_aHA_IP_rep1 |
| Sample type |
SRA |
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| Source name |
S2
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2
genotype: S2-WT
treatment: 40 uM CuSO4
antibody: anti-HA
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| Treatment protocol |
Transcription factor expression was induced by adding CuSO4 to the cell culture media. For all induction experiments, cells were plated at 1×10^6 cells per mL. Experiments were performed with cells harvested 48 hours after induction.
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| Growth protocol |
Drosophila S2 cells were cultured at 27°C in Schneider's medium supplemented with 10% fetal bovine serum and antibiotic/antimycotic.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each ChIP experiment, ~50 million cells were crosslinked by adding methanol-free formaldehyde directly to the cell culture medium to a final concentration of 0.8%. Cells were rotated on a nutator for 7 minutes at room temperature (RT) before quenching of crosslinking by adding glycine to 125 mM. Crosslinked cells were pelleted by centrifugation at 600 × g for 3 minutes. Cells were washed twice with 1X PBS. Cells were lysed by resuspending in lysis buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton-X 100) with protease inhibitors (Pierce) and incubating on ice for 10 minutes. The resulting chromatin was pelleted for 5 minutes at 1500 × g and resuspended in RIPA buffer. Chromatin was sonicated in a Covaris S220 Ultrasonicator (5 cycles of 120 seconds with 60 second delay, 170 peak power, 10% duty factor, 200 cycles/burst). Sonicated chromatin was centrifuged for 10 minutes at 10,000 × g to pellet insoluble material. 5% of the supernatant was set aside as input, and the remainder was incubated at 4°C overnight with antibodies (6 µL anti-Zld, 8 µL anti-Grh, 10 µg anti-Twi, 7.5 µL anti-HA). 20 µL protein A beads (Dynabeads Protein A, ThermoFisher Scientific) blocked with BSA were added and samples were incubated at 4°C for 4 hours. Beads were separated on a magnet and washed 3× with low salt wash buffer (10 mM Tris pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton-X 100, 150 mM NaCl), 2× high salt wash buffer (10 mM Tris pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton-X 100, 300 mM NaC), 2× LiCl wash buffer (0.25 M LiCl, 0.5% NP40, 0.5% sodium deoxycholate), and 1× with TE buffer with NaCl (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 50 mM NaCl). Beads were resuspended in elution buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA) and incubated at 65°C for 10 minutes. The IP and input samples were treated with 4.5 µL RNAse A for 30 minutes. 5 µL Proteinase K was added and samples were incubated overnight at 65°C to reverse crosslinking. DNA was isolated by phenol:chloroform extraction, precipitated with EtOH, and resuspended in 20 µL H2O.
Preparation of sequencing libraries was performed using NEBNext Ultra II kit (NEB) with 7 PCR cycles for library amplification. Sequencing was performed on the Illumina Hi-Seq4000 using 50bp single-end reads, or on the Illumina NovaSeq 6000 using 150bp paired-end reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
S2-WT_aHA_IP.bw
S2-WT_aHA_IP.narrowPeak
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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.
Assembly: dm6
Supplementary files format and content: bigwig file: contains z-score normalized read depth for each sample. Reads from two biological replicates were combined prior to generation of bigWig.
Supplementary files format and content: narrowPeak files: contain MACS2 peaks for ChIP samples.
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| Submission date |
Mar 21, 2023 |
| Last update date |
May 02, 2023 |
| Contact name |
Melissa M Harrison |
| E-mail(s) |
[email protected]
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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 |
GPL21306 |
| Series (2) |
| GSE227882 |
Protein-intrinsic properties and context-dependent effects regulate pioneer-factor binding and function [ChIP-Seq] |
| GSE227884 |
Protein-intrinsic properties and context-dependent effects regulate pioneer-factor binding and function |
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| Relations |
| BioSample |
SAMN33846021 |
| SRA |
SRX19742717 |
| 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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