MINA1GFP_YA_RNAseq_1
Caenorhabditis elegans
Protocols: All C. elegans strains were maintained and raised at 20 °C on NGM agar seeded with Escherichia coli OP50 (Brenner, 1974). MINA-1::GFP animals were bleached and their progeny grown on plates for 68 h at 20°C until they reached the young adult stage. For CLIP-seq data, three biological replicates were harvested and irradiated with UV-B (3 kJ/m2) as described (Zisoulis et al, 2010). Around 200'000 worms were collected for one CLIP experiment. Irradiated and washed worm pellets were lysed by sonication in RIPA buffer (25 mM HEPES-K at pH 7.5, 100 mM KCl, 1% [v/v] NP-40, 0.25% [v/v] sodium deoxycholate, 0.1% [v/v] SDS, 0.5 mM DTT, protease inhibitor cocktail, Roche). Cleared lysates were digested with RNase T1 (Fermentas) (final concentration 1 unit / μL) for 15 min at 22°C. Subsequently, MINA-1::GFP fusion proteins were immunoprecipitated with GFP-trap beads (ChromoTek) for 1 h at 4°C. 150 μL of beads were used for one experiment. For RNA-seq data, For RNA-seq data, RNA extraction was done using TRIzol reagent (Life Technologies) in accordance with the manufacturer's protocol and additionally purified by a solution of acidic phenol:chloroform (5:1 v/v) pH 4.5 (Ambion) followed by a second chloroform extraction to obtain high-grade RNA for sequencing. Finally, DNA traces were degraded by DNAse I (Ambion). For CLIP-seq data, RNA labeling and cDNA library preparation were performed according to the PAR-CLIP protocol (Hafner et al, 2010). For RNA-seq data, libraries for poly(A)+ enriched RNA sequencing were prepared using Directional mRNA-seq sample preparation protocol from Illumina. Same adapters were used for both CLIP-Seq and RNA-Seq.
ENA-FIRST-PUBLIC
2019-01-31T17:02:08Z
ENA-LAST-UPDATE
2018-11-26T10:20:34Z
External Id
SAMEA5129448
INSDC center name
Institute of Pharmaceutical Sciences, ETH Zurich
INSDC first public
2019-01-31T17:02:08Z
INSDC last update
2018-11-26T10:20:34Z
INSDC status
public
Submitter Id
E-MTAB-7420:MINA1GFP_YA_RNAseq_1
broker name
ArrayExpress
developmental stage
adult
diet
Escherichia coli OP50
genotype
opIs408[PC41G7.3::C41G7.3(Exon17)::gfp::C41G7.3(3UTR)]; unc119(ed3)
growth condition
Nematode growth media agar plates
organism part
whole organism
sample name
E-MTAB-7420:MINA1GFP_YA_RNAseq_1
scientific_name
Caenorhabditis elegans
sex
mixed
strain
WS5249