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GEO help: Mouse over screen elements for information. |
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Status |
Public on Dec 11, 2019 |
Title |
08HT115monos2 |
Sample type |
SRA |
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Source name |
Whole worm
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Organism |
Caenorhabditis elegans |
Characteristics |
strain: HT115 tissue: Whole worm treatment: empty vector molecule subtype: monosomal RNA
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Treatment protocol |
Worms: For RNAi knock down experiments, 5000 synchronized eggs were plated on NGMi (containing 2 mM IPTG) with a bacterial lawn of either E. coli HT115 (RNAi control strain, containing an empty vector) or mrps-5 RNAi bacteria. Mice: C57BL/6J germ-free mice were treated for two weeks with either amoxicillin (50 mg/kg/d), doxycycline (50 mg/kg/d), or a high dose doxycycline (500 mg/kg/d) in the drinking water supplemented with sucrose. Mice were sacrificed after 16 hours of fasting, and the livers were harvested and snap frozen in liquid N2.
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Growth protocol |
Worms: worms were cultured at 20°C on nematode growth medium (NGM) agar plates seeded with OP50 strain Escherichia coli as described before (Molenaars et al., 2018). Synchronized worms were harvested and snap frozen at L4 larval stage for mRNA isolation. Mice: C57BL/6J germ-free mice were used.
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Extracted molecule |
total RNA |
Extraction protocol |
For isolation of total mRNA, whole worms or liver tissue were homogenized with a 5 mm steel bead using a TissueLyser II (Qiagen) for 5 min at frequency of 30 times/sec in the presence of TRIzol (Invitrogen), then the isolation was continued according to manufacturer’s protocol. Polysomal fractions from two experiments were pooled and mRNA was extracted using TRIzol LS (Invitrogen) according to the manufacturer’s protocol. Contaminating genomic DNA was removed using RNase-Free DNase (Qiagen) and samples were cleaned up with the RNeasy MinElute Cleanup Kit (Qiagen). The NEBNext Ultra 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 Directional RNA Library Prep Kit for Illumina" (NEB #E7420). Briefly, rRNA was depleted from total RNA using the RiboZero kit. After fragmentation of the rRNA reduced RNA, a cDNA synthesis was performed. This was used for ligation with the sequencing adapters and PCR amplicification of the resulting product.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 4000 |
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Description |
R08_HT115_mono
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Data processing |
Reads were subjected to quality control FastQC (Andrews, 2010) trimmed using Trimmomatic v0.32 (Bolger et al., 2014) and aligned to either the C. elegans (worm) or M. musculus (mouse) genomes obtained from Ensembl, wbcel235.v91 and GRCm38v87, respectively, using HISAT2 v2.0.4 (Kim et al., 2015). Counts were obtained using HTSeq (v0.6.1, default parameters) (Anders et al., 2015) using the corresponding GTF taking into account the directions of the reads. Statistical analyses were performed using the edgeR (Robinson et al., 2010) and limma/voom (Ritchie et al., 2015) R packages. All genes with no counts in any of the samples were removed whilst genes with more than 2 count-per-million reads (CPM) in at least 4 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 (Robinson et al., 2010) and precision weighted using voom (Law et al., 2014), available as a supplementary table with the publication. Genome_build: wbcel235.v91 (worm) or GRCm38v87 (mouse) 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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Submission date |
Nov 02, 2018 |
Last update date |
Dec 11, 2019 |
Contact name |
Georges Janssens |
E-mail(s) |
g.e.janssens@amc.uva.nl
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Organization name |
Amsterdam UMC
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Street address |
Meibergdreef 9
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City |
Amsterdam |
ZIP/Postal code |
1105 AZ |
Country |
Netherlands |
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Platform ID |
GPL22765 |
Series (1) |
GSE122097 |
RNA-seq data for: A conserved mito-cytoplasmic translational balance links two longevity pathways |
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Relations |
BioSample |
SAMN10364877 |
SRA |
SRX4967259 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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