title: GSM5002141: TrkA-C Saline #4; Mus musculus; RNA-Seq
library_construction_protocol: We performed a RNA sequencing analysis of ipsilateral lumbar DRGs (L4-6) from WT-Vehicle, WT-MoArthritis, TrkA/C-KI Vehicle and TrkA/C-KI MoArthritis. n=4 biological sample (n=6 DRGs from 2 mice from the same group pooled per biological sample). Mice were terminally anesthetised and ipsilateral L4-L5-L6 DRGs were quickly harvested, snap frozen in liquid nitrogen and stored at -80°C until use. Total RNA of lumbar DRGs was extracted using RNeasy Micro Kit (Qiagen) according to the manufacturer's protocol. RNA integrity was analysed by a parallel capillary electrophoresis instrument (Fragment Analyser, Agilent®) and sent to Montpellier GenomiX platform (MGX, http://www.mgx.cnrs.fr) for RNA sequencing. RNA-Seq libraries were constructed with the Universal Plus mRNAseq kit from Nugen. 150ng of total RNA were used for the construction of the libraries. Using poly-T oligo attached magnetic beads, total RNA was purified in order to obtain only poly-A mRNA. Following purification, the mRNA is fragmented into small pieces under elevated temperature. The cleaved RNA fragments are reverse-transcribed into first strand cDNA using random hexamer primers, in presence of Actinomycin D. Then, the second strand cDNA was synthesized. These dscDNA had their ends repaired and adapters ligation was performed. The strand that will be sequenced was selected, and then the final products were purified and enriched with 15 cycles of PCR. The final cDNA libraries were validated with a Fragment Analyzer (Agilent) and quantified with a KAPA qPCR kit (Kapa Biosystems, Wilmington, MA). On 3 sequencing lanes of a V4 flowcell, 6 or 7 of the 20 libraries were pooled in equal proportions per lane, denatured with NaOH and diluted to 22 pM before clustering. Cluster formation, primer hybridization and single-end read 50 cycles sequencing were performed on cBot and HiSeq2500 (Illumina, San Diego, CA) respectively.