A pre-mRNA degradation pathway that selectively targets intron-containing genes requires the nuclear poly(A)-binding protein
A pre-mRNA degradation pathway that selectively targets intron-containing genes requires the nuclear poly(A)-binding protein
Total RNA was isolated from S. pombe cells using a hot phenol method followed by phenol-chloroform extractions, and precipitation as described (Lyne et al., 2003, BMC Genomics 4, 27.). 10ug total RNA was used as starting material for strand-specific RNA-seq library preparation using an unreleased early version provided by Illumina of the Illumina TruSeq Small RNA Sample Prep Kit. Briefly: poly(A)+ RNA was enriched by two rounds of poly(dT) Sera-Mag magnetic beads purification. Enriched poly(A)+ RNA was then fragmented to an average size of 200nt. Fragmented RNA was 3' de-phosphorylated with Antartic phosphatase and 5' phosphorylated with polynucleotide kinase this treatment prepares RNA fragments for subsequent ligation of Illumina RNA adaptors to their 5' and 3' ends using a 3' RNA ligase and a T4 RNA ligase, respectively. First strand cDNA was produced using a primer specific for the Illumina 3' adaptor. The library was finally amplified with 15 cycles of PCR using primers specific for the Illumina adaptors and purified using SPRI-beads (Agencourt, Beckman Coulter). Libraries size distributions and concentrations were determined on an Agilent Bioanalyzer
Experimental Factor: STRAIN_OR_LINE
FBY106
Experimental Factor: GENOTYPE
h+ ade6-M216 leu1-32 ura4-D18 his3-D1