Illumina NovaSeq 6000 paired end sequencing; Perturbation of spinal cord gene expression accompany peripheral neuropathy in murine model of type 1 diabetes
Perturbation of spinal cord gene expression accompany peripheral neuropathy in murine model of type 1 diabetes
Mice were sacrificed with a mixture of ketamine (300mg/kg, Narketan®, Vétoquinol Biowet, Poland) and xylazine (30mg/kg, VetaXyl®, Vet-Agro, Poland). Lumbar spinal cord were collected and snap‐frozen in liquid nitrogen and stored at −80°C for further analysis. Total RNA was extracted from lumbar spinal cord neuromere harvested from 32-weeks-old mice. RNA was isolated using RNeasy® Plus Universal Mini Kit and QIAzol® Lysis Reagent (both Qiagen, USA), according to manufacturer's recommendations. Subsequently, RNA integrity (RIN), quality and quantity were evaluated with microcapillary electrophoresis (2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA, USA). Only samples with RIN above 8 were used for further analysis.  "For library construction, DNA/RNA is extracted from a sample. After performing quality control (QC), qualified samples proceed to library construction. The sequencing library is prepared by random fragmentation of the DNA or cDNA sample, followed by 5' and 3' adapter ligation. Alternatively, \""tagmentation\"" combines the fragmentation and ligation reactions into a single step that greatly increases the efficiency of the library preparation process. Adapter-ligated fragments are then PCR amplified and gel purified."