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| Status |
Public on Nov 23, 2023 |
| Title |
Tollrm9rm10 2to4h CUT&Tag Rpb3 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 (2-4 h AEL)
tissue: embryos
genotype: Tollrm9/rm10 mutant
antibody: rabbit anti-Rpb3 (gift of John Lis)
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| Treatment protocol |
yw;PCNA-eGFP (60-80 min (nc 7-9), 1.5-2 h (nc 11-13) and 2-4 h AEL (nc14)), hand-sorted according to the developmental observed from the nuclear GFP signal, and Toll mutant embryos (2-4 h AEL) were dechorionated in dilute bleach, rinsed thoroughly with embryo wash buffer (PBS, 0.1% Triton X-100) and nuclei immediately extracted for CUT&Tag.
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| Growth protocol |
yw;PCNA-eGFP (wt) and 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 stages.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nuclear extraction was performed as described by 'Hainer, S.J., and Fazzio, T.G. (2019). High-Resolution Chromatin Profiling Using CUT&RUN. Curr Protoc Mol Biol 126, e85.' and CUT&TAG was performed essentially as described by 'Kaya-Okur, H.S., Wu, S.J., Codomo, C.A., Pledger, E.S., Bryson, T.D., Henikoff, J.G., Ahmad, K., and Henikoff, S. (2019). CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 10, 1930.'. Nuclear extracts were prepared using a glass douncer and loose pestle in Nuclear Extraction buffer (20 mM HEPES pH 7.9, 10 mM KCl, 0.5 mM spermidine, 0.1% Triton X-100, 20% glycerol with protease inhibitor cocktail (Roche)) and centrifuged at 700 g for 10 min at 4°C. The nuclear pellets were resuspended in Nuclear Extraction buffer. Nuclei corresponding to 50 embryos per reaction (2-4 h AEL), 100 embryos (1.5-2 h AEL) or 200 embryos (60-80 min AEL) were incubated with 30 μl of BioMag®Plus Concanavalin A beads (Polysciences) (prepared in Binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1mM CaCl2, and 1 mM MnCl2)) on a nutator for 10 min at 4°C. Nuclei-bead complexes were resuspended in 100 μl Antibody buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.05% digitonin, 2 mM EDTA pH 8.0 and 0.1% BSA supplemented with protease inhibitor cocktail (Roche)). For Toll mutant CUT&Tag, 1 μl of rabbit anti-BRD4/fs(1)h, rabbit anti-CycT, rabbit anti-Cdk9, rabbit anti-Rpb3 (a kind gift of John Lis), rabbit anti-RNA Polymerase II CTD repeat YSPTSPS (phosphor S5) (5SerP) (Abcam, ab5131) and rabbit anti-RNA Polymerase II CTD repeat YSPTSPS (phospho S2) (2SerP) (Abcam, ab5095) overnight at 4°C. For yw; PCNA-eGFP CUT&Tag, 1 μl of rabbit anti-BRD4/fs(1)h, rabbit anti-Cdk9 and rabbit anti-CBP. Following overnight incubation, tagmentation was performed using pA-Tn5 (Protein Science Facility, KI, Stockholm).
Tagmented DNA was PCR amplified using custom i5 and i7 PCR primers and Phusion® High-Fidelity PCR Master Mix with GC Buffer (NEB). PCR conditions were: 72°C for 5 min, 98°C for 30 s, followed by thermocycling (98°C for 10 s and 63°C for 10 s) for 13 cycles and final extension at 72°C for 1 min. Amplified libraries were purified using Agencourt AMPure XP beads (Beckman Coulter) (1.1:1 bead to sample volume ratio). Libraries were pair-end (2 x 37 bp) sequenced on an Illumina NextSeq 550 platform at the BEA core facility, Karolinska Institutet, Stockholm.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
Sequenced genomic DNA tagged by Tn5
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| Data processing |
CUT&Tag-seq reads from each sequenced library were mapped to the Drosophila melanogaster (dm6) genome assembly using Bowtie2 with the default parameters . RPKM normalized CUT&Tag-seq coverage tracks were generated using the deepTools (v.3.5.1) package (default parameters with binSize = 2 (bases), normalizeUsing RPKM). The low read counts obtained from sequencing for the CUT&Tag samples 'gd7 2to4h CUT&Tag CycT Replicate 2', 'gd7 2to4h CUT&Tag BRD4 Replicate 2' and Toll10B 2to4h CUT&Tag 2SerP Replicate 2’ indicated these reactions had failed so they were excluded from the subsequent analysis .
Assembly: dm6
Supplementary files format and content: bigwigs for coverage
Library strategy: CUT&Tag
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| Submission date |
Aug 16, 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 |
GPL22106 |
| Series (2) |
| GSE211220 |
Tissue-specific RNA Polymerase II promoter-proximal pause release and burst kinetics in a Drosophila embryonic patterning network |
| GSE211383 |
Tissue-specific RNA Polymerase II promoter-proximal pause release and burst kinetics in a Drosophila embryonic patterning network [CUT&TAG] |
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| Relations |
| BioSample |
SAMN30349122 |
| SRA |
SRX17104677 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6468244_Tollrm9rm10_2to4h_CUT_Tag_Rpb3_R2.bw |
82.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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