title: 8 months old Mus musculus Transcriptome or Gene expression
design: Total RNA was prepared for small RNA Sequencing-by-Synthesis according to the prescribed procedure and standards of the Illumina Sample Preparation Protocol. The samples were quantified and equalized so that equivalent amounts of RNA from mutant and wild-type were analyzed. In brief, total RNA was purified by electrophoretic separation on a 15% TBE-urea denaturing PAGE gel and small RNA regions corresponding to the 18-30 nucleotide bands in the marker lane were excised and recovered. The 18-30 nt small RNAs were 5' and 3' RNA adapter-ligated by T4 RNA ligase and at each step length validated and purified by urea PAGE gel electrophoretic separation. The adapter-ligated small RNA was subsequently transcribed into cDNA by SuperScript II Reverse Transcriptase (Invitrogen) and PCR amplified, using primers that anneal to the ends of the adapters. The amplified cDNA constructs, too, were purified and recovered. The final quality of the library was ensured by validation of the size, purity and concentration of the cDNA library on an Agilent Technologies 2100 Bioanalyzer.