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Status |
Public on Jul 06, 2021 |
Title |
079_CTRL |
Sample type |
SRA |
|
|
Source name |
whole worm
|
Organism |
Caenorhabditis elegans |
Characteristics |
tissue: whole worm strain: N2 age: day 1 adult treatment: water vehicle
|
Treatment protocol |
~1000 worms were transferred to plates containing compounds, as well as 5-fluorouracil at larval stage 4 of development. They were treated with atracurium or water control for 24 hours before harvest. At time of harvest, worms were washed in buffer and water, then snap frozen in liquid nitrogen.
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Growth protocol |
Gravid adult worms were age-synchronized using alkaline hypochlorite treatment, and incubated in M9 buffer overnight. Larval stage 1 worms were seeded to NGM plates and grown to larval stage 4.
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Extracted molecule |
total RNA |
Extraction protocol |
For isolation of total mRNA, whole worms were homogenized with a 5 mm steel bead using a TissueLyser II (QIAGEN) for 5 min at frequency of 30 times/second. RNA was extracted according to the instructions of the RNaesy Mini Kit (QIAGEN). Contaminating genomic DNA was removed using RNase-Free DNase (QIAGEN). RNA was quantified with a NanoDrop 2000 spectrophotometer (Thermo Scientific; Breda, The Netherlands) and stored at -80°C until use. RNA libraries were prepared and sequenced with the Illumina platform by Genome Scan (Leiden, The Netherlands). The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina was used to process the sample(s). The sample preparation was performed according to the protocol "NEBNext Ultra II Directional RNA Library Prep Kit for Illumina" (NEB #E7760S/L). Briefly, mRNA was isolated from total RNA using the oligo-dT magnetic beads. After fragmentation of the mRNA, cDNA synthesis was performed. This was used for ligation with the sequencing adapters and PCR amplification of the resulting product. The quality and yield after sample preparation was measured with the Fragment Analyzer. The size of the resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp). Clustering and DNA sequencing using the NovaSeq6000 was performed according to manufacturer's protocols. A concentration of 1.1 nM of DNA was used. NovaSeq control software NCS v1.6 was used.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 4000 |
|
|
Description |
H2O_1
|
Data processing |
Reads were subjected to quality control FastQC, trimmed using Trimmomatic v0.32 and aligned to the C. elegans genome obtained from Ensembl (wbcel235.v91), using HISAT2 v2.1.0 Counts were obtained using HTSeq (v0.11.0, default parameters) using the corresponding GTF taking into account the directions of the reads. Statistical analyses were performed using the edgeR v3.26.8 and limma/voom v 3.40.6 R packages All genes with more than 2 counts in at least 3 of the samples were kept. Count data were transformed to log2-counts per million (logCPM), normalized by applying the trimmed mean of M-values method and precision weighted using voom. Genome_build: wbcel235.v91 Supplementary_files_format_and_content: csv file of CMPs (counts per million) for RNAseq data mapped to gene level for each sample
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|
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Submission date |
May 06, 2020 |
Last update date |
Jul 06, 2021 |
Contact name |
Georges E. Janssens |
E-mail(s) |
g.e.janssens@amsterdamumc.nl
|
Organization name |
Amsterdam UMC
|
Department |
Laboratory Genetic Metabolic Diseases
|
Street address |
Meibergdreef 9
|
City |
Amsterdam |
ZIP/Postal code |
1105 AZ |
Country |
Netherlands |
|
|
Platform ID |
GPL22765 |
Series (1) |
GSE149944 |
Inhibition of the neuromuscular acetylcholine receptor with atracurium activates FOXO/DAF-16-induced longevity (RNA-seq) |
|
Relations |
BioSample |
SAMN14841266 |
SRA |
SRX8264329 |