title: Illumina Genome Analyzer IIx sequencing; CLIP-seq analysis of C.elegans MINA-1::GFP transgenic animals.
design: CLIP-seq analysis of C.elegans MINA-1::GFP transgenic animals.
library_construction_protocol: 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.
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: input DNA