TITLE: Single-Nuclei Isolation From Snap Frozen Axolotl Brain with Injected EdU
AUTHORS: Ashley  Maynard, Fides  Zenk

[DESCRIPTION]
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).

[STEPS]
SECTION: Nuclei isolation
2. Use pre-cooled buffers and storeon ice, perform isolation steps on ice, use pre-cooled micro-centrifuge at 4 °C.

SECTION: Nuclei isolation
3. Put tissue in cold 1.5 mLtube

SECTION: Nuclei isolation
4. Add 50 µLof lysis buffer

SECTION: Nuclei isolation
5. Using an electric grinder. Grind the tissue for 10 s  (or 2-5 pulses depending on if the tissue persists) in the tube. Rinse the pestle with 150 µL wash buffer.

SECTION: Nuclei isolation
6. Optional: Check an aliquot of the nuclei on the Evos or at the Nikon
SECTION: Nuclei isolation
7. Spin down 5 min at 500 x g (at 4 °C)

SECTION: Nuclei isolation
8. Optional: Keep the supernatant and check an aliquot on the Evos or at the Nikon
SECTION: Nuclei isolation
9. Wash the pellet with 200 µL of wash buffer (do not disturb the pellet for optimal recovery)

SECTION: Nuclei isolation
10. Spin down at 500 x g for 5 min (at 4 °C)

SECTION: Nuclei isolation
11. Resuspend the pellet in 55 µL of wash/resuspension buffer

SECTION: Nuclei isolation
12. Count with typan blue (5 µLsample + 5 µLtrypan)

SECTION: Prepare Buffers
1. Prepare the buffers as described in the Materials section. Store buffers at 4 °C or on ice.

SECTION: Stain for EdU
13. Add 500 µL Click-iT® reaction cocktail to nuclei, mix well, and incubate at Room temperature for 30 min protected from light
SECTION: Stain for EdU
14. Wash the nuclei once with 3 mLof 1% BSA in PBS.

SECTION: Stain for EdU
15. Spin down at 500 x g for 5 min (at 4 °C)

SECTION: Stain for EdU
16. Remove the supernatant. Dislodge the pellet and resuspend the nuclei in 500 µL of Wash/Resuspension Buffer, add 5 µL DAPI and FACS immediately.
SECTION: FACS of cells
17. 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.