Right trigeminal ganglion from male WKY rat #11
Left and right TG were isolated, immediately frozen in liquid nitrogen, and stored at -80C for subsequent RNA sequencing. TG samples were homogenized using TissueRupter disposable probes (Qiagen, Cat# 990890) affixed to a PowerGen 125 homogenizer (Fisher, Cat# FS-PG125). RNA extraction was performed with the Qiagen RNeasy Mini Kit (Cat# 74104) and samples were DNase treated with the Qiagen RNase-Free DNase Set (Cat# 79256). RNA was eluted in a total volume of 50 l RNase-free H2O post extraction.  Total RNA was quality checked on a Tapestation 4200 (Agilent) and the concentration checked with a Qubit 4.0 (Life Technologies).  Based on the Qubit concentrations, the total RNA input was normalized across all samples in the project, using 200 ng from each sample. mRNA (PolyA) selection was performed with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs, #E7490L), and library construction was performed with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, #E7760L).  Libraries were evaluated on the Tapestation 4200 (Agilent) for average size and Qubit 4.0 (Life Technologies) for concentration.  Libraries were then pooled equimolar and sequenced on an Illumina NovaSeq X Plus.