|
| Status |
Public on Mar 12, 2024 |
| Title |
2C_EMB_DP_AID_EtOH_biol_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
embryo
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
tissue: embryo
cell line: MHE229 (Drosha Pasha intestinal degron)
cell type: 2-cell embryos
genotype: drsh-1(luc82[myc::AID::3XFLAG::4xGGSG::drsh-1::4xGGSG::3xFLAG::AID::myc])pash-1(luc71[pash-1::2xGGSG::3xFLAG::AID::myc]) I; reSi12 [ges-1p::TIR1::F2A::mTagBFP2::AID*::NLS::tbb-2 3'UTR] (II:0.77)
treatment: EtOH
|
| Treatment protocol |
To induce the degradation of DROSHA and PASHA in C. elegans intestine, we used the AID degron system. Mothers were treated with Auxin at L3 stages and embryos or hatched L1 were collected for the RNA-seq experiment. 250 mM auxin stock solution was prepared in ethanol and stored at 4 °C. Auxin plates or ethanol plates were prepared by the addition of auxin or only ethanol to NGM plates (final concentration: 500 µM auxin, 0.2% ethanol for auxin plates and 0.2% ethanol for ethanol plates). Plates were seeded with OP50 Escherichia coli.
|
| Growth protocol |
Strains were maintained at 20°C, using standard methods (Brenner, 1974). Bristol N2 was the wild-type strain used.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To obtain synchronized non-starved L1, we used the NemaSync C. elegans synchronizer model 5000. L1 larvae were frozen in dry ice with TRIzol Reagent (Invitrogen), and after six repetitions of freeze and thaw, RNA was extracted using the Direct-zol RNA Microprep kit (ZymoResearch, R2062). The small RNA fragments (17 to 200 nucleotides) were separated from the large RNAs (>200 nucleotides) using the Quick-RNA MicroPrep kit (ZymoResearch, R1051). The large RNA was DNAse treated using the reagent provided along with the Quick-RNA Microprep kit. Using these size separation kits, we were able to sequence both the small RNA and the mRNA coming from the same samples. An Agilent 2200 TapeStation System was used to evaluate the RIN indexes of the large RNAs (>200 nucleotides), and only samples with RNA integrity number RIN > 8 were used for RNA-seq. For 2-cell embryo RNA extraction, precisely staged 2-cell embryos were obtained by alkaline hypochlorite treatment of synchronized gravid adult hermaphrodites, and fifty 2-cell embryos were collected using a mouth pipet. Embryos were lysed for 10min at 65˚C and 1min at 85˚C in a lysis solution containing Tris pH8 5mM final, EDTA 0.25mM final, Triton 100X 0.5%, Tween 20 0.5%, Proteinase K 0.5% and Water. The lysates were subjected to RNA purification using the Quick-RNA MicroPrep kit (ZymoResearch, R1051), and small RNAs (RNA fraction from 17 to 200 nucleotides) and mRNAs (RNA fraction >200 nucleotides) were collected separately. The RNA fraction > 200 nucleotides were used for preparing RNA-seq libraries.
DNase-treated total RNA with RIN > 8 was used to prepare strand-specific RNA libraries. Ribosomal and mitochondrial rRNAs were depleted using a custom RNAse-H-based method to degrade rRNAs using complementary oligos as described in42.Strand-specific RNA libraries were prepared using at least 100 ng of rRNA-depleted RNAs using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760S). RNA libraries were analysed on Agilent 2200 TapeStation System using high sensitivity D1000 screentapes and quantified using the Qubit Fluorometer High Sensitivity dsDNA assay kit (Thermo Fisher Scientific, Q32851). Multiplexed libraries were sequenced on a NextSeq 2000 Illumina platform.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
| |
|
| Description |
RNA-seq from 2-cell embryos
|
| Data processing |
For all of the library, normalized bigwig files were generated from the mapping results using millions of non-structural mappers as a normalization factor. This normalized coverage information was computed for 10 bp bins using the bedtoolsand bedopsv.2.4.26 packages.
Assembly: ce11 C. elegans Sequencing Consortium WBcel235
Supplementary files format and content: normalized big wig files will be provided for each libraries
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| |
|
| Submission date |
Mar 11, 2024 |
| Last update date |
Mar 12, 2024 |
| Contact name |
Germano Cecere |
| E-mail(s) |
germano.cecere@pasteur.fr
|
| Phone |
0033140613225
|
| Organization name |
Institut Pasteur
|
| Department |
Development and stem cell biology
|
| Lab |
Mechanisms of Epigenetic Inheritance
|
| Street address |
Institut Pasteur, 28 Rue Du Docteur Roux, Batiment Monod, 4eme Etage
|
| City |
Paris |
| ZIP/Postal code |
75724 |
| Country |
France |
| |
|
| Platform ID |
GPL32326 |
| Series (1) |
| GSE261341 |
Soma-to-Germline miRNA inheritance via Yolk Promotes Stress Resilience in Progeny [mRNA] |
|
| Relations |
| BioSample |
SAMN40379326 |
| SRA |
SRX23901810 |
