|
| Status |
Public on Dec 10, 2019 |
| Title |
piwi_CRISPR_line1_F12_RNA-seq |
| Sample type |
SRA |
| |
|
| Source name |
nematodes
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
genotype: CRISPR-cas9 piwi mutant line 1
tissue: whole worm
developmental stage: young adult, 48 hours post hatching
treatment: none
|
| Treatment protocol |
RNAi treatment was performed using worms grown on 15 cm Petri dishes seeded with concentrated RNAi food. The Arhinger RNAi library was used to pick the selected bacteria clones.
|
| 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. 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 was used for downstream applications. For the preparation of total RNA extracted from generation F4 of wild-type and piwi mutant worms 10 manually picked worms were used.
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. The sequences of the oligos used are provided in supplementary table S3. 1 µg of DNase-treated total RNA was mixed with 1 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 2.0.4) with default parameters
Mapped reads were used to estimate the abundance of protein coding genes using featureCounts (version 1.5.2) 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 2.4.26) 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 |
Jan 24, 2019 |
| Last update date |
Dec 10, 2019 |
| 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) |
| GSE125599 |
Small RNA-mediated transgenerational silencing of histone genes impairs fertility in piRNA mutants (RNA-Seq) |
| GSE125601 |
Small RNA-mediated transgenerational silencing of histone genes impairs fertility in piRNA mutants |
|
| Relations |
| BioSample |
SAMN10812963 |
| SRA |
SRX5287743 |
