Apr 02, 2022
Single-Nuclei Isolation From Snap Frozen Axolotl Brain with Injected EdU
This protocol is a draft, published without a DOI.
- 1ETHZ - ETH Zurich
Protocol Citation: Ashley Maynard, Fides Zenk 2022. Single-Nuclei Isolation From Snap Frozen Axolotl Brain with Injected EdU. protocols.io https://protocols.io/view/single-nuclei-isolation-from-snap-frozen-axolotl-b-b6yqrfvw
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: 60144
Keywords: Nuclei isolation, Axolotl, Pallium, EdU, Div-seq
Abstract
This protocol enables isolation of single nuclei with EdU incorporation from frozen 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), Div-Seq (DOI: 10.1126/science.aad7038), and EdU FACs protocol. 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).
EdU staining was performed immediately using Click-iT EdU Flow Cytometry assay Kit (Thermo Fisher Scientific, #C10424), 500 µl reaction buffer was added directly to the resuspension buffer (mix is made following the manufacturer’s protocol), mixed well and left in RT for 30min. 3ml of wash buffer was added to the resuspended nuclei and mixed well, then nuclei were spun down for 5 min at 500xg (4°C), supernatant was removed and nuclei were resuspended in 500 µl PBS + 0.5% BSA with DAPI and FACS sorted immediately (sorted for DAPI+/EdU+). FACS 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:
| A | B | C | D | |
| Reagent | [Stock] | [Final] | Volume | |
| Tri-HCL (pH 7.4) | 1 M | 10mM | 60ul | |
| NaCl | 5M | 10 mM | 12ul | |
| MgCl2 | 1M | 3mM | 18ul | |
| BSA | 10% | 1% | 600ul | |
| RNase Inhibitor | M0314S 40000U/ul (dilute 1:100 first = 400U/ul [working]) | 1U/ul | 15ul | |
| Nuclease-free Water | 5.295ml |
Volume will make 6mL
Div-Seq Lysis Buffer:
| A | B | C | D | |
| Reagent | [Stock] | [Final] | Volume | |
| Tri-HCL (pH 7.4) | 1 M | 10mM | 100ul | |
| NaCl | 5M | 10 mM | 20ul | |
| MgCl2 | 1M | 3mM | 30ul | |
| Tween-20 | 10% | 0.01% | 10ul | |
| NP-40 | 10% | 0.01% | 20ul | |
| Digitonin (dissolve at 65C) | 5% | 0.001% | 2ul | |
| BSA | 10% | 1% | 1000ul | |
| DTT | 1M | 1mM | 10ul | |
| RNase Inhibitor | M0314S 40000U/ul | 1U/ul | 2.5ul | |
| Roche Protease Inhibitor | 100x (x1 tablet in 500ul of water) cOmplete, EDTA-free Protease Inhibiotr Cocktail 11873580001 Roche | 1x | 100ul | |
| Nuclease-free Water | 8.8ml |
Volume will make 10mL
Prepare Click-iT® EdU reagents
- To make a 10X stock solution of the Click-iT® EdU buffer additive (Component G), add 2 mL of deionized water to the vial and mix until the Click-iT® EdU buffer additive is fully dissolved. After use, store any remaining stock solution at ≤–20˚C. When stored as directed, the stock solution is stable for up to 1 year.
- Prepare a working solution of Alexa Fluor® 647 azide (Cat. no. C10424 by adding 130 µl of DMSO to Component B and mix well. After use, store any remaining working solution at ≤–20°C. When stored as directed, this working solution is stable for up to 1 year
- Prepare 1X Click-iT® EdU buffer additive by diluting the 10X stock solution 1:10 in deionized water (2 reactions = 10ul + 90ul)
Prepare the Click-iT® reaction cocktail (according to number of reactions needed):
| A | B | C | D | |
| Reaction component | Number of reactions | |||
| x1 | x2 | x3 | ||
| PBS | 438 µl | 875 µl | 1.314 mL | |
| CuSO4 (Component F) | 10 µl | 20 µl | 30 µl | |
| Fluorescent dye azide | 2.5 µl | 5 µl | 7.5 µl | |
| Reaction Buffer Additive | 50 µl | 100 µl | 150 µl | |
| Total reaction volume | 500 µl | 1 mL | 1.5 mL | |
Prepare Buffers
Prepare Buffers
Prepare the buffers as described in the Materials section. Store buffers at 4 °C or On ice .
Nuclei isolation
Nuclei isolation
10m 10s
10m 10s
Use pre-cooled buffers and storeOn ice , perform isolation steps On ice , use pre-cooled micro-centrifuge at 4 °C .
Put tissue in cold 1.5 mL tube
Add 50 µL of lysis buffer
Using an electric grinder. Grind the tissue for 00:00:10 (or 2-5 pulses depending on if the tissue persists) in the tube. Rinse the pestle with 150 µL wash buffer.
10s
Optional: Check an aliquot of the nuclei on the Evos or at the Nikon
Spin down 00:05:00 at 500 x g (at 4 °C )
5m
Optional: Keep the supernatant and check an aliquot on the Evos or at the Nikon
Wash the pellet with 200 µL of wash buffer (do not disturb the pellet for optimal recovery)
Spin down at 500 x g for 00:05:00 (at 4 °C )
5m
Resuspend the pellet in 55 µL of wash/resuspension buffer
Count with typan blue (5 µL sample + 5 µL trypan)
Stain for EdU
Stain for EdU
35m
35m
Add 500 µL Click-iT® reaction cocktail to nuclei, mix well, and incubate at Room temperature for 00:30:00 protected from light
30m
Wash the nuclei once with 3 mL of 1% BSA in PBS.
Spin down at 500 x g for 00:05:00 (at 4 °C )
5m
Remove the supernatant. Dislodge the pellet and resuspend the nuclei in 500 µL of Wash/Resuspension Buffer, add 5 µL DAPI and FACS immediately.
FACS of cells
FACS of cells
To obtain our EdU+ cells, FACS a negative control first (no EdU injection in sample but also stained for EdU (steps #13-16)). From this you can gate for DAPI+ and EdU+ cells.