Dissociate Protoplast Protocol

Biological materials

Young plant.

Reagents

MES (Sigma, cat. no. M8250)

Mannitol (Sigma, cat.no.M4125)

CaCl2 (Sigma, cat.no. C7902)

KCl (Sigma, cat.no. P3911)

MgCl2 (Sigma, cat.no. M9272)

NaCl (BBI, cat.no.7647-14-5)

CellulaseR10 (YakultPharmaceuticalInd. Co., Ltd., Japan)

MacerozymeR10 (YakultPharmaceuticalInd. Co., Ltd., Japan)

Bovine Serum Albumin (BSA) (Cat. No. #A8020, Solarbio, Beijing, China)

HemiCellulase (Sigma)

Equipment

Cell Strainer (40 Micrometre, cat.no.F613461-9001)

Scalpel

Petri dish

10mL or 50mL centrifuge tube and 2mL centrifuge tube

Centrifuge with adjustable acceleration

Reagent setup

Enzymolysis Solution

Water bath at 55 degree celsius for 10 minutes, then add ddH2O (0.2ml), CaCl2 (100 Micrometre) and BSA (0.01g) in sequence.

Preparation of protoplast suspension

1. Enzymatic hydrolysis of tissue samples

Plant tissue were cut into 1-2 mm cuttings. Pour the previously prepared enzymolysis solution into the petri dish, add the cut hypocotyls to the enzymolysis solution (ensure that the tissue is completely immersed in the enzymolysis solution), shake at room temperature at a low speed, and avoid light for 4 -6 hours.

2. Filtration suspension

The single cell suspension after enzymolysis was filtered with a 40 Micrometre Cell Strainer. Take the filtered cell suspension into a centrifuge tube, add twice the volume of PBS (with 0.04% BSA) and mix it in an ultra-low speed centrifuge, centrifuge at 70g for 3 minutes, discard the supernatant, repeat this step, and finally leave a certain amount of precipitate, Mix well with a cut pipette tip, filter into a new centrifuge tube with a 40 Micrometre cell strainer.

3. Microscopy for cell state

A small amount of suspension was dripped onto the slide and observe the cell number and cell viability (tested by Fluorescein Diacetate) under a microscope.

4. Reverse transcription and 10X genomics library construction

Approximately 15,000 counted cells were used for reverse transcription and subsequently library preparation according to instruction.