3p YsRNA 2 rep 2
RNY5 mutant pools were transfected into NIH 3T3 murine cells using Fugene 6. After 24 h the cells were treated with staurosporine to induce apoptosis and the generation of YsRNAs. After 8 h the cells were harvested and total RNA extracted. The small RNA libraries were generated according to Xu et al. and standard Illumina protocol with some modifications. For sRNA library construction 2 g of total RNA was used. For the generation of sRNA libraries from the 5 end of RNY5 a customized PCR primer containing the original Illumina index sequence plus fifteen additional nucleotides on the 3 end of the oligonucleotide. This modified primer had to be used in order to enrich for YsRNAs because of their low abundance compared to other sRNAs.