|
| Status |
Public on Apr 03, 2021 |
| Title |
RNA-seq WT repl4 |
| Sample type |
SRA |
| |
|
| Source name |
RNA-seq WT repl4
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: WT embryo at stage 5
tissue: embryo
genotype/variation: control (Bloomington #36303)
|
| Treatment protocol |
In order to induce RNAi mediated silencing of certain gene products we employed the Gal4-UAS system. By crossing males of a Gal4 driver line (Bloomington #7063) to females of the fly RNAi collection TRiP1 we obtained an F1 generation depleted for HP1 in the female germ line (Bloomington #33400). These females were crossed to siblings and produced embryos that were depleted of HP1 mRNA and protein. Embryos were collected on apple juice agar plates. In order to hand-stage the embryos they were submerged with halocarbon oil 27 (Sigma Aldrich H8773) to allow staging. Embryos of the correct developmental stage were then transferred individually into 50 µl of Trizol (ThermoScientific, 15596026), crashed with a tissue grinder, snap-frozen and stored at -80 °C until RNA extraction.
|
| Growth protocol |
The flies were reared under standard conditions (25 C) and transferred to cages to collect the embryos for further experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The Trizol solution was defrosted on ice filled up to 500 µl and incubated for 5 min at room temperature. After the addition of 100 µl chloroform and vigorous mixing, the samples were spun in a table top centrifuge at 4 °C, 12.000 g for 15 min in order to separate the organic and aqueous phase. The aqueous phase was transferred in to 300 µl of chloroform and the step the first step was repeated. Subsequently the aqueous phase was transferred into isopropanol containing 20 µg of glycogen. The RNA was precipitated at -20 °C at least overnight and pelleted by centrifugation (4 °C, 12.000 g for 60 min). The pellet was washed with 80% ethanol and resuspended in DNase-digestion buffer (2 U TurboDNase Ambion, 1x TurboDNase buffer Ambion). After digestion for 30 min at 37 °C the reaction was stopped by adding EDTA to a final concentration of 15 mM and heating the samples to 75 °C for 10 min. The RNA was quantified using Qubit RNA BR Assay kit (ThermoScientific Q10210) and integrity was assessed on agarose gels or the fragmentanalyzer (Agilent).
The Illumina TruSeq Stranded Total RNA protocol was used for library preparation for next generation sequencing.
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
RNA-seq WT repl4
|
| Data processing |
Reads were mapped to D. melanogaster (build dm6) using STAR [Dobin et al 2013], using the following options: --outSJfilterReads Unique --outFilterType BySJout --outFilterMultimapNmax 1000000 --alignSJoverhangMin 6 --alignSJDBoverhangMin 2 --outFilterMismatchNoverLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonicalUnannotated --seedSearchStartLmax 50 --twopassMode basic. Gene expression was quantified using qCount from QuasR package [Gaidatzis et al 2015] using the "TxDb.Dmelanogaster.UCSC.dm6.ensGene" database for gene annotation and UCSC repeatMasker table for repeats annotation
Genome_build: dm6
Supplementary_files_format_and_content: csv file containing the count table
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| |
|
| Submission date |
Nov 18, 2019 |
| Last update date |
Apr 03, 2021 |
| Contact name |
Yinxiu Zhan |
| E-mail(s) |
[email protected]
|
| Organization name |
European Institute for Oncology
|
| Street address |
Via Adamello 16
|
| City |
Milano |
| ZIP/Postal code |
20139 |
| Country |
Italy |
| |
|
| Platform ID |
GPL19132 |
| Series (2) |
| GSE140541 |
HP1 drives de novo 3D genome reorganization in early Drosophila embryos (RNA-seq) |
| GSE140542 |
HP1 drives de novo 3D genome reorganization in early Drosophila embryos |
|
| Relations |
| BioSample |
SAMN13319858 |
| SRA |
SRX7175291 |
