|
| Status |
Public on May 02, 2023 |
| Title |
S2-Grh_DMSO_aH3K27me3_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
S2
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2
genotype: S2-Grh
antibody: aH3K27me3
treatment: DMSO
|
| 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 |
CUT&RUN was performed using EpiCypher reagents according to the manufacturer protocol. 2×105 cells were used for each CUT&RUN reaction. Overnight incubation with antibodies was performed in antibody buffer containing 0.05% digitonin. 0.5 µL anti-H3K27me3 (Cell signaling technologies) or rabbit IgG were used for CUT&RUN. Prior to addition of antibody, a 1:5 dilution of K-MetStat Panel spike-in nucleosomes (EpiCypher, cat. # 19-1002) was added to each reaction.
Libraries were prepared using the NEBNext Ultra II kit (NEB). During library preparation, cleanup steps were performed using a 1.1X ratio of Axygen paramagnetic beads, as recommended by EpiCypher. PCR amplification was performed with the following conditions: 98°C for 45 seconds, then 14 cycles of 98°C for 15 seconds, 60°C for 10 seconds, and a final extension of 72°C for 1 minute.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
S2-Grh_DMSO_aH3K27me3_total.bw
|
| Data processing |
The percentage of reads containing barcoded Epicypher nucleosomes was calculated and 1 divided by this percentage was used as the scaling factor for spike-in normalization. The z-score normalized read depth was multiplied by these scaling factors to generate spike-in normalized bigWigs.
Raw CUT&RUN 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.
Peak calling was performed using MACS2 with parameters –broad -f BAMPE –keep-dup all -g dm.
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-scoore normalized read depth of merged replicates
Library strategy: CUT&RUN
|
| |
|
| 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) |
| GSE227881 |
Protein-intrinsic properties and context-dependent effects regulate pioneer-factor binding and function [CUT&RUN] |
| GSE227884 |
Protein-intrinsic properties and context-dependent effects regulate pioneer-factor binding and function |
|
| Relations |
| BioSample |
SAMN33846150 |
| SRA |
SRX19742891 |
