title: GSM8726090: CTL bridge 2, D14 following sciatic neve injury; Mus musculus; RNA-Seq
library_construction_protocol: 6 nerve fragments (each fragment had a size of 0.5mm) were combined from multiple mice for each sample. 3 independent samples were obtained for each region and genotype of interest. Samples were then crushed and homogenised on dry ice and lysed using 200µL Trizol reagent. A further 300µL of Trizol reagent was then added and incubated for 5 mins at RT. After lysis, 100µL of chloroform was added, the samples vortexed for 2 mins, and centrifuged for 15 mins at 4˚C. The upper RNA phase was transferred to a fresh tube and 70% ethanol added in a 1:1 mix (final ethanol concentration of 35%). RNA was then extracted using PureLink RNA Micro Scale kit columns (Invitrogen, 12183016) according to the manufacturer's instructions.  An additional DNA removal step was performed to eliminate genomic DNA using a RNase-free DNase kit (Qiagen), according to the manufacturer's instructions. RNA was eluted in nuclease-free H2O, and quantified by Nanodrop measurement. Ribosomal RNA (rRNA) was depleted with the Ribo-Zero Gold Kit (Illumina) and extent of rRNA depletion was analysed with Bioanalyzer RNA pico chip. The NEB Ultra Directional RNA Library Preparation Kit (NEB, E7420) was used to prepare the RNA-Seq libraries from the enriched material. All of the enriched material was used and following 15 cycles of amplification, the RNA was purified using AMPure XP beads (Beckman Coulter, A63882) The libraries were quantified using Qubit and the size distribution was analysed using the Agilent 2100 Bioanalyser. The final libraries were pooled in equimolar amounts.