GSM4852611: P2_7-NGF_cb_1_GGCTAC_L001; Rattus norvegicus; RNA-Seq
To ensure that the axons were free of cell bodies, prior to each experiment axon compartments were incubated with Hoechst 33342 (10mg/mL in PBS for 20 min at 37˚C) and observed under an inverted fluorescent microscope. Cultures showing cell nuclei in the axon compartments or leakage of the dye in the central compartment were discarded. Total axonal and cell bodies RNA was purified from the lateral compartments of 52 or 36 chambers and the central compartment of 7 or 6 chambers respectively obtained from 3 or more independent cultures. Total RNA was isolated using PureLink® RNA Micro Scale Kit, according to the manufacturer's instructions with minor modifications. Briefly, axons and cell bodies were collected from chambers using lysis buffer (300L) containing 10%mercaptoethanol. Total mRNA bound to the columns was washed and eluted twice in elution buffer (12L). Aliquots of each sample was reverse transcribed in a 20L reaction volume containing random hexamer mix and 50U SuperScript III Reverse Transcriptase at 50°C for 1 hr. To check the quality of samples and the absence of cell bodies contamination in axon samples, first-strand cDNAs (5L) were PCR amplified in a 25L PCR reaction containing actin beta or histone H4 specific primers (0.20M), dNTPs (200nM) and Go Taq polymerase (1.25U, Promega). Libraries were prepared at the Liverpool Centre for Genomic Research using the strand-specific Script-seq protocol (Illumina). Paired-end sequencing (2x 150bp) of four indexed libraries was performed on the Illumina HiSeq2000 platform, generating in excess of 80M mappable reads per sample.