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| Status |
Public on Apr 26, 2024 |
| Title |
24hAPF Brain ATAC Rep1 |
| Sample type |
SRA |
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| Source name |
brain
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: brain
Sex: female
genotype: y,w,hsflp/w ; + ; +
treatment: none
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| Treatment protocol |
Larval samples were dissected from wandering larvae isolated from uncrowded vials. Vials with more than ~100 larvae were diluted into fresh vials to keep larvae uncrowded. Pupa were collected from vials at the White Pre-pupa stage (WPP) as described (Flegel et al., 2013), which was taken as 0h After Puparium Formation (APF) and reared on damp Kimwipes at 25°C to the indicated hours APF.
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| Growth protocol |
Animals were raised at 25°C on Bloomington Cornmeal media without malt extract
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Dissect 16 eyes or 3 brains per sample in Wash+ solution (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% BSA, 0.5 mM Spermidine and Roche complete Protease Inhibitor Tablet: 1 tablet per 50 mL). Pipet 100ul of dissected tissues in Wash+ solution directly into 100µL 2X Collagenase/Dispase solution. Incubate at 23C for 30 minutes, shaking at 500 rpm. Vortex for 10 seconds at 60% max speed on a benchtop vortexer. Incubate another 10 minutes at 23C, shaking at 500 rpm. Vortex again for 10 seconds at 60% speed. From here, we use the Omni-ATAC Protocol (Corces et al., 2017). Spin down at 800 x g, 5 minutes, at 4C. Remove supernatant and wash in 200 µL 1X PBS. Repeat 4C spin and remove supernatant. Resuspend cell pellet in 50 µl cold ATAC-RSB (10mM Tris-HCl pH 7.4, 10 mM NaCl, 3mM MgCl2) supplemented with 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin. Pipette up and down 3 times. Incubate on ice 3 minutes. Add 1 mL cold ATAC-RSB containing 0.1% Tween-20 without NP40 or Digitonin. Invert tube 3 times. Spin down at 800 x g for 10 minutes, 4°C. Discard supernatant and resuspend nuclei in 50 µL transposition reaction mix: 25 µl 2x TDE1 Buffer, 3.5 µl Tn5 Enzyme, 16.5 µl PBS, 0.5 µL 1% Digitonin, 0.5 µL 10% Tween-20, 5 µL water. Incubate at 37°C for 30 minutes shaking at 1000 rpm.Purify using a Qiagen MinElute kit according to manufacturer’s protocol. Elute in 21 µl Elution Buffer (10 mM Tris buffer pH 8.0).
Library amplification was performed with NEBNext High-Fidelity 2X PCR Master Mix, according to cycling conditions described in Corces et al., 2017, and qPCR side reactions to optimize the number of PCR cycles for each sample. Libraries were assessed on an Agilent Bioanalyzer prior to sequencing
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Adaptors and low-quality bases trimmed with cutadapt version 1.18, -q 20, -m 25
Alignment to Drosophila genome dm6 with bowtie2 version 2.4.1, --local --very-sensitive -X 1000
PCR duplicates marked with Picard version 2.8.1 MarkDuplicates
Bam file generation, sub-nucleosomal fragments selected, downsampling, sorting, and indexing with samtools version 1.5
Bigwig file generation with Deeptools bamCoverage --binsize 10 --extendreads --normalizeUsing CPM --ignoreForNormalization chrM
Peaks called using py-macs2 verison 2.2.4 callpeak
Read coverage in peaks quantified by subread version 2.0.3 featurecounts
Assembly: dm6
Supplementary files format and content: BigWig
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| Submission date |
Apr 03, 2024 |
| Last update date |
Apr 26, 2024 |
| Contact name |
Elizabeth Fogarty |
| Organization name |
University of Michigan
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| Street address |
1105 N. University Ave
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
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| Platform ID |
GPL25244 |
| Series (2) |
| GSE263159 |
Transcriptional repression and enhancer decommissioning silence cell cycle genes in postmitotic tissues: ATAC-Seq Timecourse from Drosophila Eye and Brain |
| GSE263160 |
Transcriptional repression and enhancer decommissioning silence cell cycle genes in postmitotic tissues |
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| Relations |
| BioSample |
SAMN40743299 |
| SRA |
SRX24149571 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8186975_Brain_ATAC_24APF_1_NFR_CPM.bw |
54.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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