|
| Status |
Public on May 02, 2023 |
| Title |
S2-Grh_100uM_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
S2
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2
genotype: S2-Grh
treatment: 100uM CuSO4
|
| 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.
|
| Growth protocol |
Drosophila S2 cells were cultured at 27°C in Schneider's medium supplemented with 10% fetal bovine serum and antibiotic/antimycotic.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
2×10^5 cells were harvested for ATAC-seq by centrifugation at 600 × g for 3 minutes at RT. Cells were washed once in 100 µL 1X PBS and resuspended in 100 µL ATAC lysis buffer (10 mM Tris pH 7.5, 10 mM NaCl, 3 mM MgCl2,0.1% NP-40). Cells were centrifuged at 600 × g for 10 minutes at 4°C. Supernatant was removed and pellet was resuspended in 47.5 µL buffer TD (Illumina). 2.5 uL of Tagment DNA Enzyme (Illumina) was added and samples were incubated at 37°C for 30 minutes. Tagmented DNA was purified using MinElute Reaction Cleanup Kit (Qiagen) and eluted in 10 µL of buffer EB.
DNA was PCR amplified for 12 cycles with the following conditions:72°C for 5 minutes, 98°C for 30 seconds, then 12 cycles of 98°C for 10 seconds, 63°C for 30 seconds and 72°C for 1 minute. Amplified libraries were purified using a 1.2X ratio of Axygen paramagnetic beads.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
S2-Grh_100uM_small.bw
S2-Grh_ATACseq_peaks.narrowPeak
|
| Data processing |
Raw ATAC-seq reads were trimmed to remove adapter sequences using NGmerge version 0.3.
Trimmed reads were aligned to the Drosophila melanogaster genome (version dm6) using bowtie2 with paramters -k 2 --very-sensitive --no-mixed --no-discordant -X 5000.
Reads with a mapping quality < 30 and reads aligning to the mitochondrial genome or scaffolds were discarded. Only fragments < 100bp were considered for downstream analysis.
Reads were combined across all replicates and peak calling was performed using MACS2 with parameters -f BAMPE --keep-dup all -g dm --call-summits.
featureCounts was used to count the number of reads aligning within 200bp (100bp upstream or downstream) of peak summits.
Differential accessibility analysis was performed using DESeq2. Regions with an adjusted p-value < 0.05 were considered differentially accessible.
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 files: z-score normalized read depth for merged replicates. Only small fragments (< 100bp) are included.
Supplementary files format and content: narrowPeak files: MACS2 peaks called on combined replicates for all samples within each experiment.
|
| |
|
| Submission date |
Mar 21, 2023 |
| Last update date |
May 02, 2023 |
| Contact name |
Melissa M Harrison |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Wisconsin Madison
|
| Department |
Biomolecular Chemistry
|
| Lab |
1135 Biochemical Sciences Bldg
|
| Street address |
420 Henry Mall
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL25244 |
| Series (2) |
| GSE227880 |
Protein-intrinsic properties and context-dependent effects regulate pioneer-factor binding and function [ATAC-Seq] |
| GSE227884 |
Protein-intrinsic properties and context-dependent effects regulate pioneer-factor binding and function |
|
| Relations |
| BioSample |
SAMN33846484 |
| SRA |
SRX19743265 |
