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| Status |
Public on Nov 23, 2023 |
| Title |
Tollrm9/rm10 3h PRO-seq Replicate 2 |
| Sample type |
SRA |
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| Source name |
Embryos
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Tollrm9/rm10 mutant
developmental stage: Early embryonic stage (2.5-3 h AEL)
molecule subtype: Nascent RNA
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| Treatment protocol |
Naive yw;PCNA-eGFP (60-80 min AEL), hand-sorted according to the developmental observed from the nuclear GFP signal, and Toll mutant embryos (2.5-3 h and 4.5-5 h AEL) were dechorionated in dilute bleach, rinsed thoroughly with embryo wash buffer (PBS, 0.1% Triton X-100), snap frozen in liquid nitrogen and stored at -80 degrees Celsius.
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| Growth protocol |
Wild-type (from yw;PCNA-eGFP) 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 stage.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Precision Run-On sequencing (PRO-seq) and the more sensitive qPRO-seq were performed according to the papers 'Kwak, H., Fuda, N.J., Core, L.J., and Lis, J.T. (2013). Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339, 950-953.' and 'Judd, J., Wojenski, L.A., Wainman, L.M., Tippens, N.D., Rice, E.J., Dziubek, A., Villafano, G.J., Wissink, E.M., Versluis, P., Bagepalli, L., et al. (2020). A rapid, sensitive, scalable method for Precision Run-On sequencing (PRO-seq). bioRxiv, 2020.2005.2018.102277.'. Embryos were resuspended in cold nuclear extraction buffer A (10 mM Tris-HCl pH 7.5, 300 mM sucrose, 10 mM NaCl, 3 mM CaCl2, 2 mM MgCl2, 0.1% Triton X, 0.5 mM DTT, protease inhibitor cocktail (Roche) and 4 u/ml RNase inhibitor (SUPERaseIN, Ambion)), transferred to a dounce homogenizer and dounced with the loose pestle for 20 strokes. To remove large debris, the suspension was passed through mesh followed by douncing with a tight pestle for 10 strokes. Nuclei were pelleted at 700 g for 10 min at 4°C and washed twice in buffer A and once in buffer D (10 mM Tris-HCl pH 8, 25% glycerol, 5mM MgAc2, 0.1 mM EDTA, 5 mM DTT). For PRO-seq, isolated nuclei corresponding to approximately 10 million cells, and for qPRO-seq from 1 million cells, were resuspended in buffer D and stored at -80°C for subsequent nuclear run-on (NRO) reactions with biotinylated nucleotides.
Library construction was performed according to the papers 'Kwak, H., Fuda, N.J., Core, L.J., and Lis, J.T. (2013). Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339, 950-953.' and 'Judd, J., Wojenski, L.A., Wainman, L.M., Tippens, N.D., Rice, E.J., Dziubek, A., Villafano, G.J., Wissink, E.M., Versluis, P., Bagepalli, L., et al. (2020). A rapid, sensitive, scalable method for Precision Run-On sequencing (PRO-seq). bioRxiv, 2020.2005.2018.102277.'. Samples were sequenced single-end (1x75 bp) on the Illumina NextSeq 550 platform at the BEA core facility, Karolinska Institutet, Stockholm.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
Tollrm9/rm10_3h_PRO_R2
Sequenced biotinylated nuclear run-on RNA
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| Data processing |
Library strategy: PRO-seq
PRO-seq reads from each sequencing library were mapped to the Drosophila melanogaster (dm6) genome assembly using Bowtie2 with the default parameters. Strand separated RPKM normalized (bigwig) PRO-seq coverage tracks from individual replicates were generated using the deepTools (v.3.5.1) package 'bamCoverage' (default parameters with binSize = 2 (bases), normalizeUsing RPKM) . Strand separated files of the mean RPKM signal from both replicates were produced by first merging the read alignment (bam) files produced by Bowtie2 for each replicate using samtools merge (Samtools), from which bigwig files were obtained using the 'bamCoverage' tool (deepTools). In order to better visualize the signal from pausing and gene body regions simultaneously, the bin size was extended to 10 bases when producing the merged bigwig files.
Assembly: dm6
Supplementary files format and content: bigwig for coverage
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| Submission date |
Aug 14, 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) |
| GSE211180 |
Tissue-specific RNA Polymerase II promoter-proximal pause release and burst kinetics in a Drosophila embryonic patterning network [PRO-seq] |
| GSE211220 |
Tissue-specific RNA Polymerase II promoter-proximal pause release and burst kinetics in a Drosophila embryonic patterning network |
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| Relations |
| BioSample |
SAMN30306441 |
| SRA |
SRX17058446 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6454913_Tollrm9rm10_3h_PRO_R2_F.bw |
42.4 Mb |
(ftp)(http) |
BW |
| GSM6454913_Tollrm9rm10_3h_PRO_R2_R.bw |
42.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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