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| Status |
Public on Sep 11, 2023 |
| Title |
cas9_4-6h_csRNA |
| Sample type |
SRA |
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| Source name |
embryo
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Cas9
tissue: embryo
genotype: tinWT
time: 4-6h
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| Growth protocol |
F3, M6 and Cas9 embryos were aged at 25oC and collected at 0-2h, 2-4h, 4-6h and 6-8h time points.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from dechorionated embryos using TRI Reagent (Sigma-Merck) according to the manufacturer’s protocol, followed by ethanol precipitation for further purification.
csRNA-seq v. 5.2 (Duttke et al. 2019). Total RNA was heat denatured and short RNAs (18-65 nt) purified by 15% UREA-PAGE. 5% of these short RNAs are used to generate input libraries (input RNA-seq) and the remainder cap-selected with 5´ monophosphate-dependent exonuclease (TER51020) followed by two phosphatase (CIP) treatments. Sequencing libraries for input-RNAseqsmall RNA-seq (csInput) and csRNAseqcsRNA-seq were generated using the NEB sRNA kit but with addition of RppH forby decapping the RNA using RppH (Hetzel et al. 2016). ) followed by library preparation using the NEB sRNA kit.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
Sequencing data was analyzed using HOMER csRNAseq module (http://homer.ucsd.edu/homer/ngs/csRNAseq/index.html(http://homer.ucsd.edu/homer/ngs/csRNAseq/index.html)) and R custom scripts. 3′ adapter sequences of the reads were trimmed using HOMER (Heinz et al. 2010) and aligned to dm6 genome using STAR (version 2.7.10a) (Dobin et al. 2013). Reads were visualized as strand-specific bedGraph using HOMER makeUCSCfile command with -style tss parameter. Peak calling was performed using the findcsRNATSS.pl function in HOMER (Duttke et al. 2019), with inputusing the small RNA- seq used(csInput) as background to eliminate transcripts from degraded and high-abundance RNAssmall and miRNAs in csRNA-seq libraries. HOMER annotatePeaks.pl command was used with -rlog parameter for calculating the normalized expression values for each peak used in downstream analyses. It was also used for generating transcription profile plots, for example annotatePeaks.pl tss dm6 -size 400 -hist 10 -pc 3. For differential expression, getDiffExpression.pl -edgeR -simpleNorm -dispersion 0.05 -AvsA was used on raw counts. ComplexHeatmap R package (Gu et al. 2016) was used for hierarchal clustering. HOMER analyzeClusters.pl was used for motifs and GO terms enrichment analysis in the identified cluster.
Assembly: dm6
Supplementary files format and content: .bedGraph.gz contains the analyzed peaks file, separately for each strand
Supplementary files format and content: all.merged.tss.txt.gz contains all annotated TSS by all time points merged into single file
Supplementary files format and content: all.rlog.byTime.txt.gz contains the normalized rlog counts for all samples and timepoints
Library strategy: capped short RNA-seq (csRNA-seq)
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| Submission date |
Dec 28, 2022 |
| Last update date |
Sep 11, 2023 |
| Contact name |
Tamar Juven-Gershon |
| E-mail(s) |
Tamar.Gershon@biu.ac.il
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| Organization name |
Bar-Ilan University
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| Street address |
Life Sciences Building (212) Room 102
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| City |
Ramat Gan |
| ZIP/Postal code |
5290002 |
| Country |
Israel |
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| Platform ID |
GPL30203 |
| Series (1) |
| GSE221852 |
nascent RNA sequencing during embryonic development of tinWT and tinmDPE embryos |
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| Relations |
| BioSample |
SAMN32472079 |
| SRA |
SRX18865225 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6900783_cas9_4-6h.csRNA.negStrand.bedGraph.gz |
758.1 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM6900783_cas9_4-6h.csRNA.posStrand.bedGraph.gz |
673.1 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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