Description

The ALG-1 CLIP-seq tracks show the density of reads that were mapped to the C. elegans genome from independent ALG-1 CLIP-seq experiments in larval stage 4 (L4) wildtype and ALG-1 mutant worms. The regions determined to be occupied by ALG-1, as indicated by the analyses described below, are also available in the ALG-1 binding sites in larval stage 4 worms track. Genic regions were defined based on RNA-seq reads publicly available from Hillier et al., 2009. The reannotated genic regions are displayed as the subtrack "Reannotated genic regions based on data from L3 and L4 RNAseq from Hillier et al".

ALG-1 is a C. elegans Argonaute protein that mediates the interaction of miRNAs and their mRNA targets. CLIP-seq is a biochemical approach to determine RNA-protein interaction loci.

Methods

Cross-linking immunoprecipitation, followed by deep sequencing (CLIP-seq), was performed on the Argonaute protein ALG-1 in both wild-type (WT) and ALG-1 mutant (MT) L4 worms.

Reads were aligned to the C. elegans genome (ce6) using bowtie (version 0.9.9.2) from three independent CLIP-seq experiments in both WT and ALG-1 worms. Reads were computationally extended 50nt in the 3' direction to account for the expected size of PCR products that were sequenced. Biologically reproducible regions were determined based on gene-specific weighting of reads in each of the 3 experiments in the two experimental conditions (WT and MT). Reads in each of the experimental conditions were then collapsed, and biologically reproducible regions were further scrutinized for significance above background using the Poisson distribution on pre-mRNA. Regions that passed the Poisson-derived density cutoff and overlapped at least 25% of their length between WT and MT experiments were eliminated, and thus ALG-1 binding sites are defined as regions that are reproducible in independent biological experiments, contain more reads than expected by the Poisson distribution, and do not appear in MT experiments.

RNA-seq reads from Hillier et al. in L3 and L4 worms were used to extend annotated genic regions into untranslated regions.

Detailed descriptions of these methods can be found in the online supplementary materials for Zisoulis et al, 2010.

Credits

Data for this track were supplied to UCSC by the Gene Yeo lab in the Department of Cellular and Molecular Medicine, University of California, San Diego, CA.

References

Hillier LW, Reinke V, Green P, Hirst M, Marra MA, Waterston RH. Massively parallel sequencing of the polyadenylated transcriptome of C. elegans. Genome Res. 2009 Apr;19(4):657-66. PMID: 19181841; PMC: PMC2665784

Zisoulis DG, Lovci MT, Wilbert ML, Hutt KR, Liang TY, Pasquinelli AE, Yeo GW. Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans. Nat Struct Mol Biol. 2010 Feb;17(2):173-9. PMID: 20062054; PMC: PMC2834287