NextSeq 500 paired end sequencing; Overexpression of c-Jun in mouse Schwann cells of uninjured nerves
Overexpression of c-Jun in mouse Schwann cells of uninjured nerves
Schwann cell cultures were prepared from sciatic nerves essentially as in Morgan et al., (1991) and Arthur-Farraj et al., (2011). After enzymatic dissociation and centrifugation, the cell pellet was resuspended in defined medium (DM) (Meier et al., 1999), containing 10 -6 M insulin and 5% HS, and plated in drops on coverslips coated with Poly-L-lysine and laminin. Cells were incubated at 37°C/5%CO2 and allowed to adhere for 24 hr. After 24 hr, the medium was changed to DM/0.5% HS (controls), or DM with 10ng/ml NRG1 alone, or DM with NRG1 10ng/ml and dbcAMP 1mM for 48 hr before fixation and immunolabelling. Total RNA was isolated using the RNeasy Lipid Tissue Mini Kit (Qiagen) with a DNase I step performed to eliminate traces of genomic DNA. The purified mRNA was fragmented, and primed with random hexamers. Strand-specific first strand cDNA was generated using Reverse Transcriptase in the presence of Actinomycin D. The second cDNA strand was synthesised using dUTP in place of dTTP, to mark the second strand. The resultant cDNA was then “A-tailed” at the 3' end to prevent self-ligation and adapter dimerisation. Truncated adaptors, containing a T overhang were ligated to the A-Tailed cDNA. Successfully ligated cDNA molecules were then enriched with limited cycle PCR (10-14 cycles).
Experimental Factor: phenotype
6 fold c-Jun overexpression