|
| Status |
Public on Nov 20, 2021 |
| Title |
hrde1_tm1200_YA_rep2_RNA-seq |
| Sample type |
SRA |
| |
|
| Source name |
nematodes
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
genotype: hrde-1(tm1200)
tissue: whole worm
developmental stage: Young Adult
treatment: none
|
| Treatment protocol |
A COPAS Biosorter was used to obtain homogeneously staged worm populations at the indicated developmentals stages.
|
| Growth protocol |
Strains were maintained at 20°C, using standard methods. (Stiernagle, T. Maintenance of C. elegans. WormBook : the online review of C. elegans biology 1–11 (2006))
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Synchronous populations of L1 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. Worms were collected at the indicated developmental stage: early L4 ( 38 hours post hatching (hph)), late L4 (44hph) or Young Adult ( 48hph). The harvested worms were washed three times with M9 buffer and the pellet was frozen in dry ice with Trizol Reagent (Ambion). After five repetitions of freeze and thaw, total RNA was isolated according to the Trizol Reagent protocol. Ten micrograms of RNA were treated with 2 U of Turbo DNase (Ambion) at 37°C for 30 minutes followed by phenol-extraction and isopropanol precipitation. Agilent 2200 TapeStation System was used to evaluate the RIN indexes of all the RNA preps, and only samples with RIN > 8 were used for downstream applications.
DNase-treated total RNA with RIN > 8 was used to prepare strand-specific RNA libraries. We developed an RNase H based method to degrade C. elegans and mitochondrial ribosomal RNAs (rRNAs) using 50nt oligos complementary to rRNA and mtRNA C. elegans sequences. 100 ng of DNase-treated total RNA was mixed with 1.5 µg of oligos at equimolar concentration and 1X probe hybridization buffer (200 mM NaCl, 10 mM Tris pH 7.5) and incubated in a thermocycler using the following parameters: 2 minutes at 95°C followed by 0.1°C/sec at 95-45°C, and 2 minutes hold at 45°C. Next 2 µl of Thermostable RNase H (epicentre) was added to the reactions together with 1X RNase H reaction buffer 50 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgCl2) and incubated for 30 minutes at 45°C. Next, digested RNAs were treated with 2 U of Turbo DNase (Ambion) at 37°C for 30 minutes followed by purification using 2.2 volumes of Agencourt RNAClean XP Beads (Beckman Coulter, NC0068576) following the manufacturer’s instructions. 100 ng of Ribosomal-depleted RNAs were then used to generate strand-specific RNA libraries using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (E7760S). Multiplexed RNA libraries were quantified using Qubit Fluorometer High Sensitivity dsDNA assay kit (ThermoFisher, Q32851) and sequenced on NextSeq-500 Illumina platform using the NextSeq 500/550 High Output v2 kit 75 cycles (FC-404-2005).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
total RNA, rRNA-depleted
|
| Data processing |
Reads were mapped on the C. elegans genome (WBcel235) using hisat2 (version 2.1.0) with default parameters
Mapped reads were used to estimate the abundance of protein coding genes using featureCounts (version 1.6.3) with options -O -M --primary -s 2 --fracOverlap 0 and annotations corresponding to protein coding genes (as annotated in the iGenome distribution of WBcel235 obtained at ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Caenorhabditis_elegans/Ensembl/WBcel235/Caenorhabditis_elegans_Ensembl_WBcel235.tar.gz)
The alignment was used to generate the normalized bigwig file using millions of summed forward reads per kilobase in protein coding genes as normalizer. This was done with a custom bash script using bedtools (version 2.27.1), bedops (version 2.4.35) and bedGraphToBigWig (version 4)
Genome_build: C. elegans ce11 (WBcel235)
Supplementary_files_format_and_content: bigwig files allowing to display the normalized abundance of RNAs along the genome
|
| |
|
| Submission date |
Sep 01, 2020 |
| Last update date |
Nov 20, 2021 |
| 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 |
GPL19757 |
| Series (2) |
| GSE157317 |
piRNAs initiate transcriptional silencing of spermatogenic genes during C. elegans germline development [RNA-seq] |
| GSE157319 |
piRNAs initiate transcriptional silencing of spermatogenic genes during C. elegans germline development |
|
| Relations |
| BioSample |
SAMN15962328 |
| SRA |
SRX9055683 |
