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| Status |
Public on Mar 12, 2024 |
| Title |
DP_AID_Auxin_300mM_biol_rep3 |
| Sample type |
SRA |
| |
|
| Source name |
small RNAs from 2-cell embryos
|
| 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: mothers exposed to 300mM NaCl and Auxin
|
| Treatment protocol |
In all experiments using AID strains, 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. Synchronized worms were placed on auxin or ethanol plates from L3 stage. To collect embryos from day 3 adult AID strains the auxin treatment was initiated at day 1 of adulthood. For experiments using the AID2 system, 10mM stock solution of 5-Ph-IAA was prepared in ethanol and stored at -20 ˚C for not more than a month. 5-Ph-IAA plates were prepared at a final concentration of 1µM. For osmotic stress treatment, synchronized populations of L1 larvae, obtained by hypochlorite treatment of gravid adults, were grown on NGM plates for 48 hours. After 48 hours, worms were washed from the NGM plates and divided on two NGM plates seeded with OP-50 E. coli and containing 50mM NaCl (control treatment) or 300mM NaCl (mild osmotic stress). After 24 hours of exposure, adults were collected and washed from the plates. Adults were pelleted by sedimentation rather than centrifugation to avoid collecting laid eggs derived from not fully exposed worms. Eggs from adults were collected by hypochlorite treatment and 2-cell stage embryos were manually picked for small RNA sequencing.
|
| 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 |
Synchronous populations of worms were grown at 20°C on NGM plates seeded with OP50 E. coli concentrated food at a density of maximum 40,000 animals per 15 cm Petri dish and harvested at Young Adult stage at 48 hours post hatching. The harvested animals were washed three times with M9 buffer and 40 µl of worm pellet was frozen in dry ice with TRI Reagent (MRC, Inc.). After five repetitions of freeze and thaw, total RNA was isolated according to the TRI Reagent protocol. For the 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. A small RNA spike-in was added before embryo lysis.
The small RNA library preparation was performed essentially as described previously by Barucci et al., 2020.
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| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
NextSeq 2000 |
| |
|
| 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. For the 2-cell embryo small RNA sequencing, counts were normalized in each sample using spike-ins counts (scaling factor per sample = 10^3 x total_nb_spikes_counted / total_nb_reads_aligned) in a dataset. Scaling factors were then generally "minimized" : all scaling factors of the dataset were divided by the smallest scaling factor in the dataset).
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) |
| GSE261340 |
Soma-to-Germline miRNA inheritance via Yolk Promotes Stress Resilience in Progeny [small RNA] |
|
| Relations |
| BioSample |
SAMN40379279 |
| SRA |
SRX23901842 |
