Single Nucleus RNAseq Sample Prep from Nodose Ganglia

Nodose ganglia are dissected and flash frozen in liquid nitrogen.Store at -80C until ready to isolate nuclei.Grind up the sample with liquid nitrogen using the 1.5mL centrifuge tube and fitting pestle.

Add 500 ul Lysis Buffer to pulverized tissue, pipette 10x up and down and place on ice

Incubate 5 min with the overhead shaker in the cold room

Spin down 5 min 500g at 4C using soft settings. Remove supernatant

Resuspend tissue in 400 ul Sorting Buffer by pipetting 10x up and down.

Filter with a green 30 um CellTRic (Sysmex) into FACS tube. Wash filter with another 200 ul SB. Gently clap rack against bench to fully transfer the nuclei suspension. Add 6 ul DRAQ7 (Cell signaling) to stain nuclei and place sample on ice.

For collection of sorted nuclei add 50 ul Collection Buffer

to a 1.5 ml LoBind tube (collection tube).

Use 100 um Chip for sorting of nuclei

Set temperature for sample and collection to 5C

Identify single nuclei based FSC/SSC signals andDRAQ7 fluorescence in FL-6 (PE-Cy7) channel

Sort 60,000 (200 ul) nuclei (Gate: “Sorting”) with the setting “Purity” into the prepared collection tube. After sorting is finished (10-20 min), mix sample and keep on ice.

Spin down 15 min 1000xg at 4C (Using swinging bucket rotor). After centrifugation, there should be a tiny blue pellet be visible. Remove supernatant and resuspend in 18 ul RB by pipetting 10x (Recovery of nuclei should be around 50-60%). Leave sample on ice.

Check for single cell suspension and count nuclei using hemocytometer. Mix 5 ul sample + 5 ul Trypan blue and count 4 big squares.

Calculation: (counted number)/4*20=nuclei/ul). Adjust concentration to 1000 nuclei/ul. Follow 10xGenomics library prep protocol.