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Sample GSM3454854

Query DataSets for GSM3454854

Status

Public on Dec 11, 2019

Title

17mrps5monos2

Sample type

SRA

Source name

Whole worm

Organism

Caenorhabditis elegans

Characteristics

strain: HT115
tissue: Whole worm
treatment: mrps-5 RNAi
molecule subtype: monosomal RNA

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.

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.

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

R17_mrps5_mono

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

Submission date

Nov 02, 2018

Last update date

Dec 11, 2019

Contact name

Georges Janssens

E-mail(s)

g.e.janssens@amc.uva.nl

Organization name

Amsterdam UMC

Street address

Meibergdreef 9