Caenorhabditis elegans
Protocols: Worms of the following genotypes were used in this study: 1) clk-1(qm30) mutants, 2) clk-1(qm30); clk-1(nuc) mutants (rescued with a copy of clk-1 lacking the mitochondrial targeting signal and therefore localizing to the nucleus) and 3) clk-1(qm30); clk-1(WT) mutants (rescued with a wild-type copy of clk-1). The worms were cultured at 20 °C on nematode growth medium (NGM) agar plates seeded with OP50 strain Escherichia coli. Synchronized worms were harvested and snap frozen at L4 larval stage for total RNA isolation, or polysome profiling. For isolation of total RNA, worms 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) and 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). Samples were processed for Illumina using the NEBNext Ultra Directional RNA Library Prep Kit (NEB #E7420) according to manufacturer's instructions. Briefly, rRNA was depleted from total RNA using the rRNA depletion kit (NEB# E6310). After fragmentation of the rRNA reduced RNA, a cDNA synthesis was performed in order to ligate with the sequencing adapters and PCR amplification of the resulting product.
ENA-FIRST-PUBLIC
2018-08-28T17:03:21Z
ENA-LAST-UPDATE
2018-08-24T17:31:37Z
External Id
SAMEA4858553
INSDC center name
KU Leuven, Centre of Microbial and Plant Genetics (CMPG)
INSDC first public
2018-08-28T17:03:21Z
INSDC last update
2018-08-24T17:31:37Z
INSDC status
public
Submitter Id
E-MTAB-7166:clk-1 + 2
broker name
ArrayExpress
developmental stage
L4 larva
fraction
total RNA
genotype
clk-1(qm30)
sample name
E-MTAB-7166:clk-1 + 2
scientific_name
Caenorhabditis elegans
strain
MQ130