Dec 09, 2021

Public workspaceSingle Nucleus RNAseq Sample Prep from Nodose Ganglia

DOI
dx.doi.org/10.17504/protocols.io.v72e9qe
  • 1University of California, San Diego;
  • 2University of California-San Diego
  • SPARC
    Tech. support email: info@neuinfo.org
Jamie Verheyden
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DOI: dx.doi.org/10.17504/protocols.io.v72e9qe
Protocol Citation: Sebastian Preissl, Jamie Verheyden, Xin Sun 2021. Single Nucleus RNAseq Sample Prep from Nodose Ganglia. protocols.io https://dx.doi.org/10.17504/protocols.io.v72e9qe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: December 06, 2018
Last Modified: December 09, 2021
Protocol Integer ID: 18394
Keywords: single nucleus RNAseq
Abstract
How to isolate single nucei from nodose vagal ganglia neurons in preparation for 10XGenomics sequencing.
Materials
MATERIALS
Reagentliquid nitrogen
ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
ReagentBSAMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7906
ReagentRNasinPromega
ReagentPBS
ReagentTrypan Blue 100 mL STEMCELL Technologies Inc.Catalog #7050
ReagentcOmplete™, Mini Protease Inhibitor CocktailRocheCatalog #11836153001
ReagentEDTAInvitrogen - Thermo FisherCatalog #AM9261
ReagentDRAQ7Becton Dickinson (BD)Catalog #564904
Lysis buffer (LB):-> 550ul per sample
Prepare fresh and put on ice
ReagentStock concentrationFinal concentration1 sample2 samples
TritonX-10010%0.2%11 ul22 ul
Roche protease inhibitor25x1x22 ul44 ul
DTT200mM1mM2.75 ul5.5 ul
RNAsin (Promega, N211B)40 U/ul0.2U/ul2.75 ul5.5 ul
2 % BSA in PBS  512.5 ul1025 ul
Sort buffer (SB): -> 1ml per sample
Prepare fresh and put on ice
ReagentStock concentrationFinal concentration1 sample5 samples
EDTA (Life technologies)500mM1mM2 ul10 ul
RNAsin (Promega, N211B)40 U/ul0.2U/ul5 ul25 ul
2 % BSA in PBS  993 ul4.97 ml
Collection buffer (CB): -> 200ul per sample
Prepare fresh and put on ice
ReagentStock concentrationFinal concentration1 sample5 samples
RNAsin (Promega, N211B)40 U/ul1U/ul5 ul25 ul
5 % BSA in PBS  195 ul 975 ul
Reaction buffer (RB): -> 20ul per sample
Prepare fresh and put on ice
ReagentStock concentrationFinal concentration1 sample5 samples10 samples
5% BSA (Sigma, Molec boil grade, B6917)5%1%4 ul20 ul40 ul
RNAsin (Promega, N211B)40 U/ul0.2U/ul 0.25 ul1.25 ul2.5 ul
PBS   15.75 ul78.75 ul157.5 ul
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.