Summary of the work flow for our scRNA-seq experiments.

PROTOCOL:  Root Protoplast Preparation for Single-Cell RNA-Sequencing on the 10x Genomics Chromium System

Following is the protocol used for our scRNA-seq project.   This protocol is adapted from a method developed for moss protoplasting and posted on the 10x Genomics website:  https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstrated-protocol-moss-protoplast-suspension-for-single-cell-rna-sequencing

1. Arabidopsis seeds are sterilized with a solution of 30% bleach, 0.1% Triton X-100 (12-15 min), rinsed 3-4 times with sterile distilled water, and incubated for 3 days at 4C. We prefer to use 1000-2000 seeds per sample, but a smaller number (~200) is adequate if seed quantity is limited.  Note that the seed number required is also dependent on the cell types desired (e.g. if cells from developing regions (root tips) only are desired, then more seeds will be required).
2. Seeds are placed densely in a row(s) on 100 gauge nylon-mesh screens, which are on top of solidified MS Growth Media (see below) in square petri dishes (10×10 cm). [Note: The use of the nylon-mesh screens (Nitex 03-100/47 mesh (Sefar Inc)) aids in efficient collection of root tissue from the plates; the Arabidopsis roots are not able to grow through the 100 gauge mesh pores. Preparation of the nylon-mesh screens includes: cut to dimensions slightly less than the dish size, wrap in foil, autoclave, and place on the solidified sterile MS Growth Media (see below).]

MS Growth Media (Instructions to make 1 liter)

0.3% Gelrite (3g /L) (RPI 71010-52-1)

0.2g MES (Sigma M-2933)

1% sucrose (10g /L) (Sigma)

4.33g of MS salts (Caisson labs Cat#MSP01-50LT)

pH to 5.7-5.8 with KOH

3. The square dishes are oriented vertically and incubated under continuous light at 22C for approximately 4-5 days (~2-3 cm long roots).
4. On the day of protoplast isolation, an appropriate volume of the Solution A and the Enzyme Solution (see below) are prepared (4 ml of Enzyme Solution is needed for each sample).  [Note: It is important to be organized on the day of protoplast isolation, so as to minimize the length of time between the cutting of the roots to the loading of the root protoplasts onto the 10x Controller (should not exceed 90 min).]

Solution A  [Instructions to make 10 ml]

0.4M Mannitol   [5 ml of 0.8M Mannitol]

20mM MES (pH 5.7)  [1 ml of 0.2M MES (pH 5.7]

20mM KCl   [0.2 ml of 1M KCl]

10mM CaCl2   [0.1 ml of 1M CaCl2]

0.1% BSA  [0.1 ml of 10% BSA]

Distilled Water  [3.6 ml]

Enzyme Solution

To 5ml of Solution A add:

1.25% Cellulase (“ONOZUKA” R-10, Yakult)            0.0625 g /5ml

0.1% Pectolyase (P3026, Sigma-Aldrich)                 0.005 g /5ml

5. Place 4 ml Enzyme solution into a 35 mm diameter Petri dish, and place a 70 um strainer into the dish.
6. Open the dish of seedlings and decant all the water. Use scalpel blade to slice off the roots and scrape them into the strainer inside the 35 mm Petri dish.
7. Place the dish of roots on a rotating platform and allow to rotate at 85 rpm for 45-60 minutes at 25C. Agitate the roots 2-3 times during this period using forceps.
8. At the end of the rotation period, gently mix the roots once more and lift the strainer out of the solution. Use a transfer pipette with a wide bore to gently aspirate any liquid from the outside of the strainer and all of the liquid in the dish, and transfer into 15 ml round bottom centrifuge tube.
9. Centrifuge at 500g for 10 min at 22C using swinging bucket rotor. Discard supernatant.
10. Gently resuspend pellet (protoplasts) with 500 ul of Solution A (no enzyme).
11. Filter this protoplast solution through a 70 um strainer, then a 40 um strainer, then another 40 um strainer, and finally transfer solution to a 2 ml round bottom tube. Similar to step 8, a transfer pipette with a wide bore should be used and liquid from the outside each strainer should be collected at each of these filtering steps. [Note: These multiple filtering steps are important…Drop-seq is sensitive to debris in the protoplast solution.]
12. Centrifuge at 200g for 6 minutes at 22C using a fixed angle rotor. Discard supernatant carefully…do not disturb pellet.
13. Gently resuspend protoplasts with Solution A (30-50 ul).
14. Examine protoplasts under microscope to assess density, purity, and viability (we use Evans blue staining). There should be little/no cellular debris and >80% viable protoplasts.
15. Adjust protoplast concentration (by adding additional Solution A) to 700-1000 protoplasts / ul.
16. Load 3-8 ul (depending on desired target cell number; see 10x Genomics User Guide) onto 10x Genomics Controller.

Note:  The research article associated with this procedure can be found here.  Please cite this article to reference this protocol.