2 protocols
7.6 million of 20-nt tags from wild-type adult cleavage libraries were mapped to the C. elegans genome (ce6), requiring a perfect match. For tags mapped to at most 10 genomic loci, the final tag number was normalized with the loci number. Of genome-mapping tags, ~70% mapped to exons of annotated genes (RefSeq with new 3′UTR annotations), including rRNAs, ~25% mapped to intergenic regions, ~3% mapped to intronic regions of genes, and 0.5% mapped antisense to annotated genes. Genome_build: C. elegans genome (ce6)
nucleic acid library construction protocol
Worms were harvested at the appropriate stage, washed twice with M9 buffer, and frozen in liquid nitrogen. Total RNA was extracted using Tri-Reagent (Ambion), which was further purified with phenol:chloroform followed by precipitation with ethanol. Poly(A) RNA was enriched from wild-type adult C. elegans total RNA using the Oligotex mRNA kit (QIAGEN). A custom 5' RNA adapter containing a MmeI recognition site was ligated to the poly(A) RNA possessing a 5'-monophosphate, by T4 RNA ligase, and the ligated products were reverse transcribed. This cDNA was amplified and then digested with restriction enzyme MmeI, which cleaves 20 nts 3’ of the recognition site. This MmeI digested product was ligated to a 3’ double strand adaptor, amplified by PCR, gel-purified and submitted for Illumina sequencing.