title: NextSeq 500 paired end sequencing; Failures of nerve regeneration caused by aging or chronic denervation are rescued by restoring Schwann cell c-Jun
design: Failures of nerve regeneration caused by aging or chronic denervation are rescued by restoring Schwann cell c-Jun
library_construction_protocol: Female R26c-Junstopf mice carrying a lox-P flanked STOP cassette in front of a CAG promoter-driven c-Jun cDNA in the ROSA26 locus, were crossed with male P0Cre+/− mice (Feltri et al. 1999).  This generated P0-Cre+/−/R26c-Junstopff/+ mice, referred to here as c-Jun OE/+ mice. P0-Cre−/− littermates were used as controls. These two genotypes were used for the gene screening experiments Chronically denervated and uninjured nerves were pooled, 2 per n. Acutely denervated nerves were not pooled using 1 nerve per n. For short-term time points (<1 week) and crushes, the sciatic nerve was exposed and cut or crushed (at 3 rotation angles) at the sciatic notch. For chronic denervation (>1 week), the sciatic nerve was cut and the proximal stump was reflected back and sutured into muscle to prevent regeneration. The nerve distal to the injury was excised for analysis at various time points. Contralateral uninjured sciatic nerves served as controls. To examine regeneration into distal stumps, the nerve branches of the sciatic nerve were individually separated. The tibial nerve was cut and both proximal and distal stumps were reflected and sutured into muscle. Either immediately or following 10 weeks of chronic denervation, the distal tibial nerve stump was cut from the muscle and sutured to the freshly transected common peroneal nerve.  RNA was extracted using a RNeasy lipid tissue kit with an in column DNase step (Qiagen). The library was prepared using the Kapa mRNA Hyper Prep kit (Roche) with 100ng RNA and 15 cycles of PCR enrichment. The assay is (first) stranded (dUTP method).