10X Genomics suggested protocol for nuclei isolation for the 10X snRNA-Seq workflow

Below is a suggested protocol for nuclei isolation. 10X Genomics has a few demonstrated protocols for sample preparation, and we highly recommend to have a look at them as well.

Buffers

Wash buffer (WB)

Components Final concentration Volume for 1ml
Tris-HCl (1M, pH 7.4) 10 mM 10 µl
NaCl (5 M) 10 mM 2 µl
MgCl2 (1 M) 3 mM 3 µl
BSA (10 %) 1% 100 µl
Tween 20 (10 %) 0.1% 10 µl
Nuclease-free water - 875 µl
Table 1: Wash Buffer

Lysis Buffer (LB)

Components Final concentration Volume for 1ml
Wash buffer 980 µl
Nonidet P40 Substitute (10%) 0.1% 10 µl
Digitonin (1%) 0.01% 10 µl
Table 2: Lysis Buffer Prepare the lysis buffer fresh and keep it on ice!

Diluted Nuclei Buffer (DNB)

Components Final concentration Volume for 1ml
10X Genomics Nuclei Buffer (20x stock) 1x 50
Nuclease-free water 950
Table 3: Diluted Nuclei Buffer (DNB) Prepare the lysis buffer fresh and keep it on ice!

Nuclei isolation

  1. Isolate single cell suspension with an appropriate protocol, in a standard 1.5 ml tube. Start with at least 100.000 cells as input, max 1 mio cells.

  2. If cells are FACS-sorted, pre-load 5 µl of cell medium or PBS into the tube before sorting starts

  3. Centrifuge cells at 300 rcf for 5 min at 4°C. [choose the speed appropriate for your cell type]

  4. Remove supernatant. ~5 µl may remain.

  5. Add 100 µl chilled Lysis Buffer (Table 2) and gently pipette mix three times.

  6. Without further incubation, centrifuge directly at 500 rcf for 5-10 min4 at 4°C.

  7. Remove supernatant without disrupting the nuclei pellet.

  8. Add 100 µl chilled Wash Buffer (Table 1) to the tube and gently pipette mix three times. Optionally, simply flick the tube a few times; the nuclei should resuspend easily after 3-4 taps.

  9. Quality control step: Check nuclei quality under microscope. Mix 8 µl trypan blue (0.4% solution) with 2 µl of the nuclei suspension,5 and transfer the mix to a Cell Counting Chamber for counting nuclei and checking nuclei quality (under a 40x objective, at least). Nuclei should be round with intact nuclear membrane, no aggregates and no debris. For comparison see Figure 1 below. If debris are still present in the sample, a second wash can be performed at this step.

  10. Centrifuge the remaining nuclei suspension at 500 rcf for 5 min at 4°C.

  11. Remove most of the supernatant with a P100/200 pipette, then remove the remaining liquid with a 10µl pipette without touching the bottom of the tube, to avoid dislodging the nuclei pellet.

  12. Resuspend the nuclei pellet in appropriate volume of chilled Diluted Nuclei Buffer (Table 3) , targeting the desired nuclei concentration (ideal 4.000 - 8.000 nuclei/µl)

(Picture taken from 10X Userguide)

Notes

  • Expected nuclei recovery is around 25%, so please use an appropriate number of cells as input to achieve the desired number of nuclei.
  • It is advised to test different detergent concentrations. For this, lysis buffer can be diluted with wash buffer to 1:5, 1:10, 1:20 or 1:50 for example. (1:10 dilution is a good starting point)
  • A short incubation period of 3-5min can be included at this step in case cell lysis is sub-optimal.
  • Do not reduce spinning time to less than 5 minutes.
    • You can mix 5 µl trypan blue + 5 µl nuclei mix if you have sufficient starting material or only want to check nuclei quality. The more nuclei you can observe, the better you can estimate the quality.
    • Alternatively, you can add a DNA-intercalating dye such as propidium iodide to check for nuclei integrity and free floating DNA.

REFERENCES