Apr 02, 2022

Public workspaceSingle-Nuclei Isolation From Snap Frozen Axolotl Brain

This protocol is a draft, published without a DOI.
  • 1ETHZ - ETH Zurich
Ashley Maynard
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Protocol Citation: Ashley Maynard, Fides Zenk 2022. Single-Nuclei Isolation From Snap Frozen Axolotl Brain. protocols.io https://protocols.io/view/single-nuclei-isolation-from-snap-frozen-axolotl-b-b6yprfvn
Manuscript citation:
Single-cell analyses of axolotl forebrain organization, neurogenesis, and regeneration Katharina Lust, Ashley Maynard, Tomás Gomes, Jonas Simon Fleck, J. Gray Camp, Elly M. Tanaka, Barbara Treutlein bioRxiv 2022.03.21.485045; doi: https://doi.org/10.1101/2022.03.21.485045
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: March 31, 2022
Last Modified: April 02, 2022
Protocol Integer ID: 60143
Keywords: Nuclei isolation, Axolotl, Pallium
Abstract
This protocol enables isolation of single nuclei from frozen pallium microdissections and whole pallium dissections (from axolotl) for the purpose of generating single-nuclei gene-expression libraries following a modified protocol from 10x (Demonstrated protocol CG000365, Rev B). In brief, we prepared and precooled wash and lysis buffers (see Materials). Lysis buffer was added to the sample and dissociated via short pulses with an electric grinder. The pestle of the grinder was washed with a wash buffer before centrifugation. Supernatant was removed and the pellet gently washed. After a final centrifugation the supernatant was removed and the pellet was resuspended in PBS + BSA. Resulting nuclei were then assessed (count and viability) using Trypan Blue assay, counted using the automated cell counter Countess (Thermo Fisher). The resulting nuclei are then ready for downstream analysis, including but not limited to 10x Genomics gene expression (single-nuclei RNA sequencing).
Materials
For this protocol you will need an electric grinder with pestle, we recommend:
  • Kimble 749521-1500 Polypropylene Pellet Pestle Only, 1.5mL Capacity (Case of 100)
  • Kimble Pellet Pestles 749540-0000 Drive Unit Cordless Motor with Two AA Batteries

Wash/Resuspension Buffer:

ABCD
Reagent[Stock][Final]Volume
Tri-HCL (pH 7.4)1 M10mM60ul
NaCl5M10 mM12ul
MgCl21M3mM18ul
BSA10%1%600ul
RNase InhibitorM0314S 40000U/ul (dilute 1:100 first = 400U/ul [working])1U/ul15ul
Nuclease-free Water5.295ml
Volume will make 6mL

Lysis Buffer:
ABCD
Reagent[Stock][Final]Volume
Tri-HCL (pH 7.4)1 M10mM100ul
NaCl5M10 mM20ul
MgCl21M3mM30ul
Tween-2010%0.01%10ul
NP-4010%0.01%20ul
BSA10%1%1000ul
DTT1M1mM10ul
RNase InhibitorM0314S 40000U/ul1U/ul2.5ul
Roche Protease Inhibitor100x (x1 tablet in 500ul of water) cOmplete, EDTA-free Protease Inhibiotr Cocktail 11873580001 Roche1x100ul
Nuclease-free Water8.82ml
Volume will make 10mL


Prepare Buffers
Prepare Buffers
Prepare the buffers as described in the Materials section. Store buffers at Temperature4 °C or TemperatureOn ice .

Nuclei isolation
Nuclei isolation
10m 10s
10m 10s
Use pre-cooled buffers and storeTemperatureOn ice , perform isolation steps TemperatureOn ice , use pre-cooled micro-centrifuge at Temperature4 °C .

Put tissue in cold Amount1.5 mL tube

Add Amount50 µL of lysis buffer

Using an electric grinder. Grind the tissue for Duration00:00:10 (or 2-5 pulses depending on if the tissue persists) in the tube. Rinse the pestle with Amount150 µL wash buffer.

10s
Optional: Check an aliquot of the nuclei on the Evos or at the Nikon
Optional
Spin down Duration00:05:00 at Centrifigation500 x g (at Temperature4 °C )

5m
Optional: Keep the supernatant and check an aliquot on the Evos or at the Nikon
Optional
Wash the pellet with Amount200 µL of wash buffer (do not disturb the pellet for optimal recovery)

Critical
Spin down at Centrifigation500 x g for Duration00:05:00 (at Temperature4 °C )

5m
Resuspend the pellet in Amount50 µL of wash/resuspension buffer

Count with typan blue (Amount5 µL sample + Amount5 µL trypan)