GSM1399294: pos-1 (RNAi) rep2; Caenorhabditis elegans; OTHER
Total RNA was extracted from snap frozen cell pellets Trizol extraction. 1 microgram of this was input into the library preperation. Library construction was by the PAT-seq approach. Breifly this involves a modified the ePAT approach to tagging adenylated RNA to generate libraries suitable for deep sequencing.  Briefly, adenylated RNA is sequence specifically extended by dNTPs using Klenow polymerase and an annealed DNA anchor oligonucleotide. This takes advantage of the native function of DNA polymerase to extend an RNA primer from a DNA template in second strand synthesis. Importantly, any unwanted priming to internal poly(A)-tracts in RNA is avoided by a requirement for 3’ extension in subsequent fragment selection and reverse transcription. No ribosome depletion is necessary. Here, the anchor sequence was compatible with the Illumina index primers and included a 5’ biotin moiety to facilitate handling. In a second step, the 3’ tagged RNA was subject to limited fragmentation by RNase T1. This cleaves RNA after G-residues and ensures that cleavage is only possible within the body of the RNA, not the poly(A)-tract or the DNA sequence of the extended tag. The fragmented RNA was 5’ phosphorylated to allow RNA Ligase 2 mediated ligation of an Illumina compatible splinted-linker to the RNA fragments. Reverse transcription was primed from the anchor sequence. Note: All manipulations after limited fragmentation were performed in association with streptavidin magnetic beads. The cDNA PAT-seq libraries was eluted from beads, size-selected by Urea PAGE and amplified with primers that introduce the features for directional Illumina sequencing and indexing. In samples analysed here, the window of selection was between 120-300 bases. This size range was selected to allow for ≥ 25 bases of 3’UTR sequence to map reads to the genome, the an average yeast poly(A)-tail of ~25 bases (maximum ~90 bases), the majority of reads would contain heterogeneous 5’ sequence of sufficient length to map uniquely to the yeast genome. Note: all reads run in 5’ to 3’ direction from unique sequence into a variable length of poly(A) homopolymers. This means that color balance is preserved and that any low fidelity within the homopolymers is limited to the end of the read. Cluster Generation: 9pM of libraries per lane using Illumina c-bot. Illumina protocol 15006165 Rev J, July 2012 Sequencing chemistry: 1 x 150bp sequencing using Illumina protocol 15035788 Rev A, Oct 2012
GEO
GSM1399294
GEO Accession
GSM1399294