Researchers Detail Single-Cell RNA Protocol for Arabidopsis Roots

Researchers at various institutions examining plant single-cell genomics have detailed a comprehensive single-cell RNA sequencing protocol and methodology in Nature, outlining precise laboratory workflows for isolating protoplasts and generating cellular atlases from Arabidopsis root tips. The study documents specific methods across multiple ecotypes and mutants including Col-0, Ws-2, Cvi-0, c24, Ler, aba2-3, pyl11458, and scr-3 to analyze cell lineage behavior under controlled growth conditions.

Protoplast Isolation and Growth Conditions

Seeds from wild-type ecotypes and mutants were sterilized and densely sown in rows on 1× or 0.5× MS basal medium with vitamins from Austratec on 0.9% agar plates supplemented with 1% sucrose at pH 5.7. Plates were stratified in the dark at 4 degrees Celsius for two days before growing in long-day conditions consisting of 16 hours of light and 8 hours of dark at 22 degrees Celsius. Root tips up to approximately 3 millimeters were collected at 6 to 7 days after germination for protoplast isolation using sharp tweezers.

For hard-agar water-deficit assays, seedlings were grown on 0.5× MS medium under control conditions with 100% water content or on medium with water content reduced to 50% or 20% of control levels. Additional root growth assessments involved cutting solid medium in half with a razor blade under sterile conditions to let cotyledons develop at the surface while roots penetrated and grew through the agar for homogeneous environmental exposure.

Sequencing and Quality Filtering Workflow

Root tips were placed in 15 milliliters of protoplasting solution containing 1.25% weight-to-volume cellulase, 0.3% weight-to-volume macerozyme, 0.4 molar D-mannitol, 20 millimolar MES at pH 5.7, 20 millimolar KCl, 10 millimolar CaCl2, 0.5% weight-to-volume BSA, and 5 millimolar beta-mercaptoethanol in ultrapure water. The solution was incubated at room temperature for one hour on a tube rotator and filtered through 70-micrometer and 40-micrometer cell strainers from Falcon to separate protoplasts from undissociated tissue. Cells were centrifuged at 500g for five minutes in a swinging bucket centrifuge, resuspended in resuspension buffer, and filtered through a 35-micrometer cell strainer.

Libraries were generated using reagents from a 10x Genomics Single Cell 3 Prime Library kit with a Chromium Controller and sequenced on an Illumina NextSeq 500 or NovaSeq 6000. Reads were mapped using the CellRanger pipeline version 7.1.0 to the TAIR10 Arabidopsis genome. Low-quality cells were removed using a Seurat version 5.0.0 filtering approach that isolated background barcodes from high-quality cells, typically eliminating 8 to 30% of CellRanger-identified cells.