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| Status |
Public on Nov 23, 2023 |
| Title |
Toll10B 5h ATAC Replicate 2 |
| Sample type |
SRA |
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| Source name |
embryos
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| Organism |
Drosophila melanogaster |
| Characteristics |
developmental stage: Early embryonic stage (5 h AEL)
tissue: embryos
genotype: Toll10B mutant
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| Treatment protocol |
Toll mutant embryos were collected for 0.5 h and aged accordingly to achieve three developmental time points: 2.5-3 h, 3.5-4 h and 4.5-5 h AEL. For each time point, 10 embryos per replicate presenting the correct morphology for the developmental stage sought were immediately hand-sorted.
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| Growth protocol |
Toll mutant (from gd7, Tollrm9/rm10 and Toll10B) embryos were collected from flies kept on potato-mash agar food and maintained at 25 degrees Celsius. Embryos were collected on apple juice plates with fresh yeast and aged at 25 degrees Celsius until they reached the range of hours after egg laying (AEL) for the required developmental stage.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Hand-sorted embryos were dechorionated in dilute bleach, rinsed thoroughly in embryo wash buffer (PBS, 0.1% Triton X-100) and crude nuclear extracts isolated by homogenizing the embryos using a motor pestle in ATAC lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630) and centrifugating at 700 g for 10 min. The nuclear pellet was resuspended in 22.5 μl of ATAC lysis buffer, 2.5 μl Tn5 (Tagment DNA Enzyme 1 (TDE) (Illumina)) and 25 μl Tagment DNA Buffer (Illumina) and subjected to tagmentation at 37°C on a thermomixer at 1,000 rpm. Transposition was blocked by the addition of 1% SDS and DNA purified with Agencourt AMPure XP beads (Beckman Coulter, A63881) according to the manufacturer’s instructions using a 2:1 ratio of beads to sample.
Libraries were prepared as previously described in 'Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y., and Greenleaf, W.J. (2013). Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome positions. Nat Methods 10, 1213-1218.'. Briefly, tagmented DNA was PCR amplified using 1x Phusion® High-Fidelity PCR Master Mix with GC Buffer (NEB) and 1.25 μM i5 and i7 PCR primers (Nextera® Index Kit (Illumina)) with the following PCR amplification conditions: 72°C for 5 min, followed by 10 cycles of 98°C for 10 seconds, 65°C for 1 min and 15 seconds, then 72°C for 1 min. Amplified libraries were purified with Agencourt AMPure XP beads with a 1.5:1 ratio of bead to sample volume. Libraries prepared from biological triplicates were sequenced paired-end (2 x 150 bp) on the Illumina NovaSeq platform at SciLifeLab, Stockholm. ATAC-seq reads from each sequenced library were mapped to the Drosophila melanogaster (dm6) genome assembly using Bowtie2 with the default parameters. RPKM normalized ATAC-seq coverage tracks were generated using the deepTools (v.3.5.1) package. Bigwig files of the mean RPKM were produced using the deepTools 'bigwigCompare' tool.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Sequenced genomic DNA tagged by Tn5
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| Data processing |
ATAC-seq reads from each sequenced library were mapped to the Drosophila melanogaster (dm6) genome assembly using Bowtie2 with the default parameters. RPKM normalized ATAC-seq coverage tracks were generated using the deepTools (v.3.5.1) package (default parameters with binSize = 2 (bases), normalizeUsing RPKM). Bigwig files of the mean RPKM were produced using the deepTools 'bigwigCompare' tool.
Assembly: dm6
Supplementary files format and content: bigwigs for coverage
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| Submission date |
Aug 19, 2022 |
| Last update date |
Nov 23, 2023 |
| Contact name |
Mattias Mannervik |
| E-mail(s) |
mattias.mannervik@su.se
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| Organization name |
Stockholm University
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| Department |
Dept. of Molecular Biosciences
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| Street address |
Svante Arrhenius väg 20C
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| City |
Stockholm |
| ZIP/Postal code |
106 91 |
| Country |
Sweden |
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| Platform ID |
GPL25244 |
| Series (2) |
| GSE211220 |
Tissue-specific RNA Polymerase II promoter-proximal pause release and burst kinetics in a Drosophila embryonic patterning network |
| GSE211663 |
Tissue-specific RNA Polymerase II promoter-proximal pause release and burst kinetics in a Drosophila embryonic patterning network [ATAC-Seq] |
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| Relations |
| BioSample |
SAMN30415411 |
| SRA |
SRX17155376 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6482261_Toll10B_5h_ATAC_R2.bw |
230.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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