title: GSM9284682: Sample 2 of P-PTEN KO Vagus nerve 2 weeks after HFD; Mus musculus; RNA-Seq
library_construction_protocol: Animals were euthanized with CO2 prior to vagus nerve dissection to obtain left and right cervical vagus nerves that contain the nodose ganglion. Vagus nerve samples were immediately flash frozen into 1.5 mL eppendorf tubes containing dry ice. The nerve tissue was then homogenized in nuclei extraction buffer using glass douncers.  The nuclei was then ultracentrifuged at 1000xG for ~2 hrs and then resuspended with the process repeated for 3 cycles. Samples were then filtered and washed with a sucrose column and placed in nuclei suspension buffer. Nuclei is then sorted with FACS with trypan blue to sort for cell viability. The appropriate amount of volume was used for ~7500 reads. Library preparation was performed per manafacturers instructions using 10x Genomics 3' v3 Reagent Kit and 10x Cell Ranger software Version 3.  Samples were loaded into a 10x Transportation reaction and incubated into  a 10x chip to generate a collection of gel beads in emulsions. The sample is then processed through a reverse transcription reaction to generate a shared 10x barcode containing the cDNA and all the nuclei which is then subjected to illumina sequencing.