title: 454 sequencing of mouse brain ncRNA library
design: RNA libraries were generated according to the previously described protocol (Huttenhofer and Vogel 2006). Briefly, after ultracentrifugation of total protein extracts on a 10-30% glycerol gradient, 28 fractions were collected, and pooled 4 by 4, but always excluding the 2 first (bottom) and the 2 last (top of the gradient) fractions, which were expected to contain rRNA contaminations and various RNA degradation products, respectively. Samples were twice extracted with phenol-chloroform, ethanol precipitated, and poly(C)-tailed employing poly(A) polymerase from yeast (Epicentre, Madison, WI, USA). C-tailed RNAs were ligated to a 19-nt long 5´ linker (5´-GTC AGC AAT CCC TAA C GAG) by T4 RNA ligase. RNAs were subsequently converted into cDNAs by Reverse Transcription (RT) using an anchor primer (5´-AGG AGC CAT CGT ATG TCG GGG GGG GH), amplified by PCR as described, employing complementary primers to linkers (forward, 5´-GCC TCC CTC GCG CCA TCA GGT CAG CAA TCC CTAA CGA G; reverse, 5´-GCC TTG CCA GCC CGC TCA GAG GAG CCA TCG TAT GTC G) and submitted to pyrrosequencing (GS-FLX system, Roche, GATC company, Konstanz, Germany).