|
| Status |
Public on May 29, 2024 |
| Title |
WT h3k9me3 |
| Sample type |
SRA |
| |
|
| Source name |
Ovary tissue
|
| Organism |
Drosophila melanogaster |
| Characteristics |
developmental stage: Differentiated stages
tissue: Ovary tissue
genotype: WT
chip antibody: H3K9me3 ab (Active motif 39062)
|
| Treatment protocol |
None
|
| Growth protocol |
Ovaries dissected in 1x PBS.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
PBS was removed and the samples were permeabilized in 1mL of Permeabilization Solution (PBST+1% Triton-X) rotating in RT for 1 hour. Samples were then incubated overnight at 4°C in primary antibody dilutions in freshly prepared BBT+ buffer (PBST + 1% BSA + 0.5 mM Spermidine + 2 mM EDTA + 1 large Roche complete EDTA-free tablets). Primary antibody was replaced with BBT+ buffer and quickly washed twice. Samples were then incubated in ~700 ng/ml of pAG-MNase in BBT+ buffer rotating for 4 hours at 25°C. Samples were then quickly washed twice in wash+ buffer (20 mM HEPES pH7.5 + 150 mM NaCl + 0.1% BSA + 0.5 mM Spermidine + 1 large Roche complete EDTA-free tablets in water). Samples were resuspended in 150 μl Wash+C (wash+ + 100 mM CaCl2) and incubated for 45 minutes on nutator at 4°C. The cleavage reaction was terminated by addition of 150 μl StopR (NaCl final 200 mM + EDTA final 20 mM + 100μg/mL RNaseA) and incubating the sample at 37˚C for 30 minutes. Samples were then centrifuged at 16,000 x g for 5 minutes and 300 μl of the supernatant was collected for DNA discovery. To the supernatant, 2 μL 10% SDS and 2.5 μL of 20 mg /mL Proteinase K was added and incubated at 50°C for 2 hours. Half of this was kept as a backup and half was used in bead cleanup. 20 μL AmpureXP bead slurry and 280 μL MXP buffer (20% PEG8000 + 2.5 M NaCl + 10 mM MgCl2 in water) was added to the sample and mixed thoroughly followed by 15 minutes incubation at RT. The beads were separated by magnet and supernatant was discarded. The beads were carefully washed with 80% ethanol for 30 seconds, while on the magnetic stand and air dried for 2 minutes. The beads were then resuspended in 10 μL DNase free water.
The samples from CUT&RUN assay were used for library preparation using NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (E7645, E7103) and adaptor ligated DNA were prepared without size selection.
The samples from CUT&RUN assay were used for library preparation using NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (E7645, E7103) and adaptor ligated DNA were prepared without size selection.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Reads were evaluated for their quality using FastQC v0.11.8
Reads were trimmed for adaptor sequences using Trim Galore! v0.6.6
Reads were aligned to the dm6 reference genome version for drosophila melanogaster using Bowtie2 v2.2.8 with parameters -q -I 50 -X 700 --very-sensitive-local --local --no-mixed --no-unal --no-discordant
Binary alignment maps (BAM) files were generated with samtools v1.9
MACS2 v2.1.0 was used to call significant peaks for samples BamRNAi Stwl, WT Stwl and WT BEAF. IgG was used as control to call peaks. Peaks within ENCODE blacklisted regions and repetitive sequences larger than 100 bases were removed
BigWig files were generated from BAM files using deepTools v3.2.1 bamCoverage function with parameters– normalize using RPKM–bin size 10
Assembly: dm6
Supplementary files format and content: bigWig, bed (for BamRNAi Stwl, WT Stwl and WT BEAF)
Library strategy: CUT&RUN
|
| |
|
| Submission date |
Dec 15, 2023 |
| Last update date |
May 29, 2024 |
| Contact name |
BiNGS Core |
| E-mail(s) |
[email protected]
|
| Organization name |
Icahn School of Medicine at Mount Sinai
|
| Street address |
1470 Madison Ave
|
| City |
New York |
| ZIP/Postal code |
10029 |
| Country |
USA |
| |
|
| Platform ID |
GPL25244 |
| Series (2) |
| GSE250350 |
Genome organization regulates nuclear pore complex formation and promotes differentiation during Drosophila oogenesis [CUT&RUN] |
| GSE250351 |
Genome organization regulates nuclear pore complex formation and promotes differentiation during Drosophila oogenesis |
|
| Relations |
| BioSample |
SAMN38859339 |
| SRA |
SRX22906612 |
