title: GSM9056516: Neuron rep2; Mus musculus; RNA-Seq
library_name: GSM9056516
library_construction_protocol: Naïve TRPV1cre::tdTomatofl/WT mice were euthanized, and their dorsal root ganglia (DRG) were immediately transferred into DMEM (Corning, 10 013 CV) supplemented with 50 U ml−1 penicillin, 50 μg ml−1 streptomycin (Corning, MT 3001 Cl) and 10 % fetal bovine serum (Seradigm, 3100). The tissue was dissociated for 80 min at 37 °C in HEPES buffered saline (Sigma, 51558) containing 1 mg ml−1 collagenase IV (Sigma, C0130) and 2.4 U ml−1 dispase II (Sigma, 04942078001). After digestion, ganglia were gently triturated through glass Pasteur pipettes of decreasing diameter, centrifuged through a 10 % BSA cushion and plated onto laminin coated (Sigma, L2020) culture dishes. Approximately 4 × 10^4 neurons per dish were maintained for 48 h in Neurobasal A medium (Gibco, 21103 049) supplemented with 0.05 ng μl−1  nerve growth factor (Life Technologies, 13257 019), 0.002 ng μl−1 glial cell line derived neurotrophic factor (PeproTech, 450 51 10), 0.01 mM cytosine β D arabinoside (Sigma, C6645), 200 mM L glutamine (VWR, 02 0131) and B 27 (Gibco, 17504044). Neurons were then co-cultured at a 1:10 ratio with mEERL derived small extracellular vesicles, cytotoxic CD8+ T-cells or both, each condition prepared in T cell medium supplemented with the same concentrations of NGF and GDNF to support neuronal viability. After 48 h, cells were detached with a scraper, washed in flow cytometry buffer (PBS containing 2 % FBS and 1 mM EDTA), passed through a 70 μm filter, and TRPV1+ neurons (FSChigh SSChigh tdTomatohigh) were isolated by fluorescence activated cell sorting directly into TRIzol. RNA extraction, library construction and sequencing were performed at the IRIC Genomics Platform (Université de Montréal). RNA integrity was confirmed with an Agilent Bioanalyzer (RIN > 7.5). Poly adenylated transcripts were captured and libraries prepared with the KAPA mRNA HyperPrep Kit (Kapa Biosystems, KR1352). Bar coded libraries were sequenced on an Illumina NextSeq 500 using 75 cycle single end reads.