RNA-seq of 4 week old Wistar-Kyoto Rattus norvegicus: right stellate ganglion
Rat and ganglia were cleaned and de-sheathed in dPBS without Ca2+ and Mg2+ and each sample was prepared to contain RNA from one stellate. Stellate ganglia were transferred immediately to RLT plus lysis buffer (Qiagen) with -mercaptoethanol (1%); finely chopped and vortexed for 30 s. Rat RNA was extracted using the RNeasy Micro Plus RNA Extraction Kit (Qiagen) and human RNA was extracted using the RNeasy Maxi RNA Extraction Kit (Qiagen) in accordance with the manufacturers instructions. RNA samples were snap frozen in liquid nitrogen and stored at -80 C. The RNA quality and integrity from each sample, was confirmed using a 2100 Bioanalyzer Instrument with an RNA 6000 Pico Kit (Agilent). Rat samples with an RNA integrity number (RIN). RNA extracted from the right or left stellate ganglia of four-week-old male preSHR (n=5) and age matched Wistar (n=5) was sent to the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (WTCHG) for RNA-seq library construction and sequencing using an Illumina HiSeq 4000 (Illumina, Inc., San Diego, USA). The sequencing libraries were amplified using a SMARTer (first-strand synthesis) amplification protocol and prepared for paired-end sequencing (2 x 75 bp). Each sample was sequenced on three separate lanes to minimize technical error and to increase the sequencing depth (~15-25 million reads per lane). Samples were randomized and blinded to the experimenter. The number of replicates and the sequencing parameters established, were based on recommendations from WTCHG and those published by Conesa et al., 2016.