We set up this protocol in the single-cell multi-omics facility of the School of Agriculture and Biology, Shanghai Jiao Tong University, and were supported by the National Natural Science Foundation of China (Grant No. 32070608), the Shanghai Pujiang Program (Grant No. 20PJ1405800), and Shanghai Jiao Tong University (Grant Nos. Agri-X20200202, 2019TPB05).

The following protocol has been optimized for A. thaliana wild-type (WT) seeds with no fluorescence and fluorescent marker lines for the following root cell types: xylem-pole pericycle cells (J0121), lateral root initial cells, lateral root cap cells (J3411), endodermis and cortex&#160;cells&#160;(J0571) (Figure 1A). All the marker lines were obtained from a commercial source (see Table of Materials), except for the lateral root initiation cell marker line, which was generated by introducing a GATA23 promoter-driven GFP construct into a wild-type Arabidopsis plant following a previously published report14.
1. Preparation of the plant material
<ol>
	<li>Sterilize the A. thaliana WT seeds and fluorescent marker line seeds by incubating the seeds in 20% bleach in a rotating incubator at room temperature for 15 min.</li>
	<li>Rinse the seeds in double-distilled water (ddH2O) three to five times. Perform this step on a sterile clean bench.</li>
	<li>Plate the WT and reporter line seeds on half-strength Murashige and Skoog (MS) medium with 0.8% agar (w/v)15. Grow the plants vertically for 5 days (16 h light at 23 &#176;C) after stratifying for 2 days at 4 &#176;C.</li>
</ol>
2. Protoplasting
<ol>
	<li>Prepare the protoplasting solutions2,5, referred to as Solution A and Solution B (see Table of Materials).
	<ol>
		<li>Prepare Solution A containing 400 mM mannitol, 0.05% BSA, 20 mM MES (pH 5.7), 10 mM CaCl2, and 20 mM KCl (see Table of Materials). Store Solution A at &#8722;20 &#176;C for up to 1 month.</li>
		<li>Prepare Solution B by adding 1% (w/v) cellulase R10, 1% (w/v) cellulase RS, 1% (w/v) hemicellulase, 0.5% (w/v) pectolyase, and 1% (w/v) macerozyme R10 in a fresh aliquot of Solution A. Store Solution B at &#8722;20 &#176;C for up to 1 month.</li>
	</ol>
	</li>
	<li>Gently thaw Solution A and Solution B on ice before beginning the experiment.</li>
	<li>Cut off the roots using a clean blade or scissors, and chop the roots into ~0.5 cm pieces. Submerge the roots in 1.5 mL of Solution B followed by gentle rotation (at approximately 18 rpm) at room temperature for 1.5-2 h.</li>
	<li>Filter the root protoplasts through the 40 &#181;m strainer mesh (see Table of Materials).</li>
	<li>Rinse the strainer mesh with 1-2 mL of Solution A.</li>
	<li>Combine the liquids of step 2.4 and step 2.5, and centrifuge at 300 x g for 5 min at 4 &#176;C. Discard the supernatant with a pipette, resuspend the cell pellet in 500-600 &#181;L of Solution A, and then place it on ice immediately.</li>
	<li>Transfer the resuspended cell solution to a new 5 mL test tube for cell sorting.</li>
</ol>
3. Fluorescence-Activated Cell Sorting (FACS)
<ol>
	<li>Switch on and finish the instrument setup steps on the sorter (see Table of Materials). Select the fluorescence channels, use a WT plant (no fluorescence) as a control to determine the baseline for autofluorescence, and adjust the sorting gate based on the fluorescence intensity and FSC/SSC singlets (Figure 2).</li>
	<li>Add 500 &#181;L of Solution A (step 2.1.1) into a 1.5 mL collection tube to prevent the cells from being damaged. Collect 2,000-3,000 cells per tube.</li>
	<li>After sorting, immediately place the samples on ice, centrifuge the collection tube containing the cells at 300 x g for 5 min at 4 &#176;C, and remove the supernatant with a pipette.</li>
	<li>Take 2 &#181;L of sorted cells, and check for fluorescence using a fluorescence microscope (see Table of Materials).</li>
	<li>Store the sorted cells at &#8722;80 &#176;C, or use them immediately for library construction (step 4).</li>
	<li>For single-cell index sorting, place the 96-well plate into the adapter. Calibrate the position of the plate so that the droplet falls in the center hole of the plate. Select single-cell sorting mode when sorting, enter the target number of sorted cells as 1, and start sorting.</li>
</ol>
4. Smart-seq2 library preparation
<ol>
	<li>As a result of the ultra-low amount of input, perform single-cell type RNA-seq library construction in a contamination-free environment. Before beginning the experiment, clean the bench with a surface decontaminant8 (see Table of Materials) and 75% ethanol.</li>
	<li>Prepare lysis buffer (mixture A) (Table 1) by combining 0.33 &#181;L of 10% Triton X-100, 0.55 &#181;L of RNase inhibitor, and 0.22 &#181;L of 0.1 M DTT (see Table of Materials).</li>
	<li>Add 1 &#181;L of mixture A into the sorted sample, and grind with a sterile pestle. The preferable sample volume is &#8804;0.5 &#181;L; use RNase-free water to make up the volume to 14 &#181;L. Transfer each single cell sample into a 0.2 mL thin-walled PCR tube.</li>
	<li>Prepare mixture B containing 0.44 &#181;L of oligo-dT30VN reverse transcription (RT) reaction primer (100 &#181;M) and 4.4 &#181;L of dNTPs (10 mM) (Table 2) (see Table of Materials).</li>
	<li>Add 4.4 &#181;L of mixture B to the 14 &#181;L of sample in each tube, pipette gently to mix the sample, and incubate the sample at 72 &#176;C for 3 min. After incubation, immediately put the samples on ice to hybridize the oligo-dT to the poly A tail.</li>
	<li>Prepare the reverse transcription reaction mixture (mixture C) (Table 3) (see Table of Materials). Add 21.6 &#181;L of mixture C to each tube containing the samples. Turn on the RT program on a common PCR instrument (Table 4).</li>
	<li>Perform the preamplification reaction on ice. Prepare mixture D by combining 44 &#181;L of 2x PCR polymerase mix and 0.88 &#181;L of IS PCR primer (10 &#181;M) (Table 5) (see Table of Materials). Add 40.8 &#181;L of mixture D to the 40 &#181;L of RT reaction product, and run the preamplification program (Table 6).</li>
	<li>Purify the preamplification reaction products using Ampure XP beads (see Table of Materials). Add 48 &#181;L of beads (0.6:1 ratio) into each sample from step 4.7, and gently mix the samples by pipetting.</li>
	<li>Incubate the samples at room temperature for 10 min. Place the 1.5 mL tubes containing the samples on a magnetic separation stand for 5 min. Carefully discard the supernatant from the samples without disturbing the beads.</li>
	<li>Wash the beads by resuspending them in 200 &#181;L of 80% ethanol, and place the samples on the magnetic separation stand (see Table of Materials) for another 3 min before discarding the ethanol-containing supernatant.</li>
	<li>Air-dry the samples for 10 min, and cover the tube to prevent contamination and cross-contamination during the air-drying.</li>
	<li>Resuspend the beads in 20 &#181;L of ddH2O, incubate the samples at room temperature for 5 min, and then place them on the magnetic separation stand for 5 min.</li>
	<li>Pipette out 18 &#181;L of the supernatant from each tube, and transfer the samples into new 1.5 mL centrifuge tubes. Use 1 &#181;L of the sample to assess the quality of the cDNA using a DNA quantification kit, determine the size distribution of each prelibrary using a fragment analyzer (see Table of Materials), and store the remaining sample at &#8722;20 &#176;C.</li>
	<li>Construct a cDNA library8 for Illumina sequencing16 from the prelibrary product of step 4.13 using a sequencing library preparation kit (see Table of Materials).</li>
	<li>Purify the libraries (from step 4.14) using the Ampure XP beads, quantify the purified libraries, and determine the size distribution of each library following step 4.13. 
	NOTE: Pool equal nanomoles of each library, ensuring that none of them have the same combination of Illumina index. Otherwise, the libraries can be pooled in a ratio based on the desired sequencing output and sequenced together on the same lane of the Illumina sequencer. Generally, sequencing each library to a depth of 4-6 GB, which yields &#62;10 million mapped reads, provides 20x-30x coverage of the Arabidopsis genome. Lower sequencing depths are also acceptable but might affect the significance of the differential expression analysis.</li>
</ol>
5. RNA-seq data analysis
<ol>
	<li>Trim the raw reads using Trim-Galore17 followed by mapping to the reference genome using hisat218 (daehwankimlab.github.io/hisat2),&#160;and remove the PCR duplicated fragments using Picard19 (broadinstitute.github.io/picard).</li>
	<li>Perform the raw count processing and subsequent analysis of the differentially expressed genes (DEG) with DESeq220 using at least three biological replicates for each sample. Perform clustering of the gene expression with the Pheatmap package, and visualize in an expression heatmap. 
	NOTE: The RPKM (reads per kilobase per million mapped reads) values of the genes and the TEs (transposable elements) were calculated with Stringtie18 (github.com/gpertea/stringtie) and visualized in a genome browser.</li>
</ol>

<ol>
	<li>Zhang, T. -. Q., Chen, Y., Liu, Y., Lin, W. -. H., Wang, J. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptome+atlas+and+chromatin+accessibility+landscape+reveal+differentiation+trajectories+in+the+rice+root.">Single-cell transcriptome atlas and chromatin accessibility landscape reveal differentiation trajectories in the rice root.</a> Nature Communications. 12 (1), 2053 (2021).</li><li>Denyer, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+developmental+trajectories+in+the+Arabidopsis+root+revealed+using+high-throughput+single-cell+RNA+sequencing.">Spatiotemporal developmental trajectories in the Arabidopsis root revealed using high-throughput single-cell RNA sequencing.</a> Developmental Cell. 48 (6), 840-852 (2019).</li><li>Liu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+landscape+of+rice+roots+at+the+single-cell+resolution.">Transcriptional landscape of rice roots at the single-cell resolution.</a> Molecular Plant. 14 (3), 384-394 (2021).</li><li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+dynamic+molecular+profiling+of+stomatal+lineage+cell+development+by+single-cell+RNA+sequencing.">Global dynamic molecular profiling of stomatal lineage cell development by single-cell RNA sequencing.</a> Molecular Plant. 13 (8), 1178-1193 (2020).</li><li>Shahan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-cell+Arabidopsis+root+atlas+reveals+developmental+trajectories+in+wild-type+and+cell+identity+mutants.">A single-cell Arabidopsis root atlas reveals developmental trajectories in wild-type and cell identity mutants.</a> Developmental Cell. 57 (4), 543-560 (2022).</li><li>Wendrich, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+transcription+factors+guide+plant+epidermal+responses+to+limiting+phosphate+conditions.">Vascular transcription factors guide plant epidermal responses to limiting phosphate conditions.</a> Science. 370 (6518), (2020).</li><li>Carter, A. D., Bonyadi, R., Gifford, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fluorescence-activated+cell+sorting+in+studying+plant+development+and+environmental+responses.">The use of fluorescence-activated cell sorting in studying plant development and environmental responses.</a> The International Journal of Developmental Biology. 57 (6-8), 545-552 (2013).</li><li>Picelli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full-length+RNA-seq+from+single+cells+using+Smart-seq2.">Full-length RNA-seq from single cells using Smart-seq2.</a> Nature Protocols. 9 (1), 171-181 (2014).</li><li>Wang, X., He, Y., Zhang, Q., Ren, X., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+comparative+analyses+of+10X+genomics+chromium+and+Smart-seq2.">Direct comparative analyses of 10X genomics chromium and Smart-seq2.</a> Genomics Proteomics Bioinformatics. 19 (2), 253-266 (2021).</li><li>Serrano-Ron, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+lateral+root+formation+through+single-cell+RNAsequencing+reveals+order+of+tissue+initiation.">Reconstruction of lateral root formation through single-cell RNAsequencing reveals order of tissue initiation.</a> Molecular Plant. 14 (8), 1362-1378 (2021).</li><li>Hashimshony, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CEL-Seq2:+Sensitive+highly-multiplexed+single-cell+RNA-Seq.">CEL-Seq2: Sensitive highly-multiplexed single-cell RNA-Seq.</a> Genome Biology. 17, 77 (2016).</li><li>Macaulay, I. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G&amp;T-seq:+Parallel+sequencing+of+single-cell+genomes+and+transcriptomes.">G&amp;T-seq: Parallel sequencing of single-cell genomes and transcriptomes.</a> Nature Methods. 12 (6), 519-522 (2015).</li><li>Angermueller, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+single-cell+sequencing+links+transcriptional+and+epigenetic+heterogeneity.">Parallel single-cell sequencing links transcriptional and epigenetic heterogeneity.</a> Nature Methods. 13 (3), 229-232 (2016).</li><li>De Rybel, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+aux/IAA28+signaling+cascade+activates+GATA23-dependent+specification+of+lateral+root+founder+cell+identity.">A novel aux/IAA28 signaling cascade activates GATA23-dependent specification of lateral root founder cell identity.</a> Current Biology. 20 (19), 1697-1706 (2010).</li><li>Duncombe, S. G., Barnes, W. J., Anderson, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+delivery+and+behavior+of+cellulose+synthases+in+Arabidopsis+thaliana+using+confocal+microscopy.">Imaging the delivery and behavior of cellulose synthases in Arabidopsis thaliana using confocal microscopy.</a> Methods in Cell Biology. 160, 201-213 (2020).</li><li>Levy, S. E., Myers, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancements+in+next-generation+sequencing.">Advancements in next-generation sequencing.</a> Annual Review of Genomics and Human Genetics. 17 (1), 95-115 (2016).</li><li>Ooi, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+full-length+single-cell+mRNA-seq+of+rare+cells.">High-throughput full-length single-cell mRNA-seq of rare cells.</a> PLoS One. 12 (11), e0188510 (2017).</li><li>Pertea, M., Kim, D., Pertea, G. M., Leek, J. T., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcript-level+expression+analysis+of+RNA-seq+experiments+with+HISAT,+StringTie+and+Ballgown.">Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown.</a> Nature Protocols. 11 (9), 1650-1667 (2016).</li><li>Tsyganov, K., Perry, A., Archer, S., Powell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAsik:+A+Pipeline+for+complete+and+reproducible+RNA-seq+analysis+that+runs+anywhere+with+speed+and+ease.">RNAsik: A Pipeline for complete and reproducible RNA-seq analysis that runs anywhere with speed and ease.</a> Journal of Open Source Software. 3, 583 (2018).</li><li>Love, M. I., Huber, W., Anders, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderated+estimation+of+fold+change+and+dispersion+for+RNA-seq+data+with+DESeq2.">Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.</a> Genome Biology. 15 (12), 550 (2014).</li><li>Kamiya, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MYB36+transcription+factor+orchestrates+Casparian+strip+formation.">The MYB36 transcription factor orchestrates Casparian strip formation.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (33), 10533-10538 (2015).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+types+of+bHLH+transcription+factor+determine+the+competence+of+the+pericycle+for+lateral+root+initiation.">Two types of bHLH transcription factor determine the competence of the pericycle for lateral root initiation.</a> Nature Plants. 7 (5), 633-643 (2021).</li><li>Haecker, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+dynamics+of+WOX+genes+mark+cell+fate+decisions+during+early+embryonic+patterning+in+Arabidopsis+thaliana.">Expression dynamics of WOX genes mark cell fate decisions during early embryonic patterning in Arabidopsis thaliana.</a> Development. 131 (3), 657-668 (2004).</li><li>Chen, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxin+overproduction+in+shoots+cannot+rescue+auxin+deficiencies+in+Arabidopsis+roots.">Auxin overproduction in shoots cannot rescue auxin deficiencies in Arabidopsis roots.</a> Plant Cell Physiol. 55 (6), 1072-1079 (2014).</li><li>Nichterwitz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microscopy+coupled+with+Smart-seq2+for+precise+spatial+transcriptomic+profiling.">Laser capture microscopy coupled with Smart-seq2 for precise spatial transcriptomic profiling.</a> Nature Communications. 7, 12139 (2016).</li><li>Long, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nurse+cell--derived+small+RNAs+define+paternal+epigenetic+inheritance+in+Arabidopsis.">Nurse cell--derived small RNAs define paternal epigenetic inheritance in Arabidopsis.</a> Science. 373 (6550), (2021).</li><li>Gutzat, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+shoot+stem+cells+display+dynamic+transcription+and+DNA+methylation+patterns.">Arabidopsis shoot stem cells display dynamic transcription and DNA methylation patterns.</a> EMBO Journal. 39 (20), e103667 (2020).</li></ol>

The authors have nothing to disclose.

The Smart-seq2-based protocol can generate reliable sequencing libraries from several hundreds of cells8. The quality of the starting material is essential for the accuracy of the transcriptome analysis. FACS is a powerful tool for preparing cells of interest, but this procedure, especially the protoplasting step, must be optimized for plant applications. Laser capture microdissection (LCM) or manual dissected cells can also be used as input25,26, so the protocol provided here can potentially be used in a variety of plant species and cell types with or without known marker genes.
Preparation of the plant material 
In plant developmental biology, FACS is usually used to isolate enriched cell populations expressing a fluorescent marker. The plants that express such a marker are protoplasted, and the protoplasts are eventually sorted into pure subpopulations based on the expression of the cell type-specific marker. Therefore, the signal of the fluorescent marker must be strong and specific. Additionally, the researcher should check what markers can be sorted in advance. The number of seeds needed depends on the level of expression, where the marker is expressed, how many cells are needed for downstream applications, and how many replicates are sorted. If the expression is in a single cell type with very few cells per plant, generally a larger number of plants are needed than if the marker is expressed in a higher number of cells. It is best to empirically determine the number of seeds required for individual marker lines. It is especially meaningful to perform preliminary experiments with each marker line to optimize the window for sorting and know how many cells will result from a fixed number of plants. If the experimental material is roots, to prevent the roots from sinking into the agar and to facilitate the cutting of clean roots, a suitably sized and autoclaved mesh can be laid on the medium-containing agar before the seed plating. Low-temperature stratification helps seed germination and development, so we suggest stratifying the seeds at 4 &#176;C for 2 days after sterilizing or plating and then growing the plants vertically.
Protoplasting and FACS 
Protoplasting and sorting 5-6 days after stratification work&#160;well. Solution A and Solution B must be filtered using a 0.22 &#181;m strainer. Additionally, storing Solution B at &#8722;20 &#176;C over longer time periods decreases the efficiency of the enzymes. Before beginning, Solution A and Solution B should be gently thawed on ice, which takes about 20 min. Solution B must not be shaken, as shaking Solution B can disrupt the enzymes and can cause excessive bubble formation. Washing the strainer mesh with Solution A multiple times can increase the number of cells obtained in step 2.5. As described in step 2.6, the entire supernatant should not be aspirated, as the protoplasts are at the bottom near the pellet and cannot be seen. Before sorting, it is best to ensure that the concentration of protoplasts is about 105-106 cells/mL to achieve better sorting efficiency. When setting up a new experiment, the same tissue from the WT plant is required as a control for setting up the sorting gate. In step 3.1, selecting the fluorescence channels (e.g.,&#160;PE and FITC) first and using a control sample to determine the major population according to SSC and FSC are recommended. Additionally, it is suggested to adjust the position of the gate by changing the voltage of the fluorescence channel to ensure that the control signal is located to the left of the gate (i.e., the negative group is located below 103). The sorting time must be limited to 30 min or less than 60 min to prevent alterations in gene expression, which may occur due to protoplasting or sorting. After sorting, a small number of sorted cells should be collected and checked for fluorescence (step 3.4). As much buffer should be removed as possible from the supernatant to avoid any impact on the downstream library construction (step 3.3).
Construction of the RNA-seq library 
For the construction of the RNA-seq library, we recommend using cells immediately after sorting to reduce the degradation of the RNA. It is not advisable to start with too many cells, as this may result in a poor-quality library due to inadequate reactions. The number of PCR cycles in step 4.7 and step 4.14 depends on the input amount of RNA/cDNA. The number of cycles can be increased when there is less input or lowered when there is more input. Therefore, it is recommended to take some of the mixed samples from step 4.7 and step 4.14 to run the real-time PCR with the same amplification procedure and to then determine the final number of cycles based on the results of the qPCR before the formal amplification of the prelibrary/library. In addition, before the purification step 4.8, the Ampure XP beads must be equilibrated at room temperature for at least 10 min and then vortexed well. The volume of beads in the purification step should not be increased above the 0.8:1 ratio. Otherwise, this will increase the carryover of primer dimers. In addition, one should avoid overdrying the beads in step 4.11 to prevent difficult resuspension. A qualified pre-library should have an average size of about 1.5-2 kb and a small amount of short (&#60;500 bp) fragments. Abnormal size distributions or small size primer/adapter dimer peaks in libraries are indicators of poor quality, and these samples should be discarded or undergo further rounds of beads purification (step 4.15) (Figure 3E-F).
Limitations 
This protocol can be applied to isolate cells from other plant tissues and other plant species. However, the cell isolation process is highly dependent on the preparation of the protoplasts. Some cell types are difficult to isolate, such as vascular cells and female sex cells, which are located in the interior of the tissue and/or are few in number. For cells for which it is difficult to prepare protoplasts, fluorescence-activated nuclei sorting (FANS)27 is an optional method that can be used. Meanwhile, using this protocol to obtain specific cell types by FACS depends on the availability of fluorescent marker lines. The lack of such marker lines limits the use of these methods in crops and horticulture plants. The application of high-throughput single-cell RNA-seq technology in crops will reveal novel cell-type-specific marker genes, which can be further used to develop cell-type marker lines and broaden the application capability of FACS-based cell-type RNA-seq and multi-omics studies.

Cells are the fundamental unit of all living organisms and perform structural and physiological functions. Although the cells in multicellular organisms show apparent synchronicity, cells of different types and individual cells present differences in their transcriptomes during development and environmental responses. High-throughput single-cell RNA sequencing (scRNA-seq) provides unprecedented power for understanding cellular heterogeneity. Applying scRNA-seq in plant sciences has contributed to successfully constructing a plant cell atlas1, has been used to identify rare cellular taxa in plant tissues2, has provided insight into the composition of cell types in plant tissues, and has been used to identify cellular identity and important functions employed during plant development and differentiation. In addition, it is possible to infer spatiotemporal developmental trajectories in plant tissues1,2,3 to discover new marker genes4 and study the functions of important transcription factors5 using scRNA-seq in order to reveal the evolutionary conservation of the same cell type in different plants3. Abiotic stresses are among the most important environmental influences on plant growth and development. By exploring the changes in the composition of cell types in plant tissues under different treatment conditions through single-cell transcriptome sequencing, one can also resolve the abiotic stress response mechanism6.
The potential for resolving transcriptional heterogeneity between cell types using scRNA sequencing depends on the cell isolation method and sequencing platform. Fluorescence-activated cell sorting (FACS) is a widely used technique for isolating a subpopulation of cells for scRNA-seq based on light scattering and the fluorescence properties of the cells. The development of fluorescent marker lines by transgenic technology has greatly improved the efficiency of cell isolation by FACS7. Conducting scRNA-seq using Smart-seq28 further enhances the ability to dissect the cellular heterogeneity. The Smart-seq2 method has good sensitivity for gene detection and can detect genes even with a low transcript input9. In addition to bulk cell type collection, modern cell sorters provide a single-cell index sorting format, allowing transcriptome analysis at single-cell resolution using Smart-seq210 or other multiplexed RNA-seq methods, such as CEL-seq211. Single-cell or cell-type sorting can be potentially used for many other downstream applications, such as parallel multi-omics studies12,13. Presented here is a robust and versatile protocol for isolating plant cell types, such as xylem-pole pericycle cells, lateral root cap cells, lateral root initial cells, cortex cells, and endodermal cells from the roots of Arabidopsis thaliana marker cell lines by FACS. The protocol further involves constructing the Smart-seq2 library for downstream transcriptome analysis.

<table><tbody><tr><td>0.22 &amp;micro;m strainer</td><td>Sorfa&amp;nbsp;</td><td>622110</td><td></td></tr><tr><td>Agar</td><td>Yeasen</td><td>70101ES76</td><td></td></tr><tr><td>Agilent fragment analyzer</td><td>Aglient</td><td>Aglient 5200</td><td></td></tr><tr><td>Agilent high-sensitivity DNA kit</td><td>Aglient</td><td>DNF-474-0500</td><td></td></tr><tr><td>Ampure XP beads</td><td>BECKMAN</td><td>A63881</td><td></td></tr><tr><td>Betaine</td><td>yuanye</td><td>S18046-100g</td><td></td></tr><tr><td>Bleach</td><td>Mr Muscle</td><td>FnBn83BK</td><td>20% (v/v) bleach</td></tr><tr><td>BSA</td><td>sigma</td><td>9048-46-8</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>yuanye</td><td>S24109-500g</td><td></td></tr><tr><td>Cellulase R10</td><td>Yakult (Japan)</td><td>9012-54-8</td><td></td></tr><tr><td>Cellulase RS</td><td>Yakult (Japan)</td><td>9012-54-8</td><td></td></tr><tr><td>Centrifuge tube (1.5 mL)</td><td>Eppendolf</td><td>30121589</td><td></td></tr><tr><td>DNase, RNase, DNA and RNA Away Surface Decontaminants</td><td>Beyotime</td><td>R0127</td><td></td></tr><tr><td>dNTPs (10 mM)</td><td>NEB</td><td>N0447S</td><td></td></tr><tr><td>DTT (0.1 M)</td><td>&lt;br/&gt; invitrogen</td><td>18090050</td><td></td></tr><tr><td>Ethanol</td><td>Sinopharm Chemical Reagent Co., Ltd</td><td>100092680</td><td></td></tr><tr><td>FACS</td><td>BD FACS Melody</td><td>BD-65745</td><td></td></tr><tr><td>FACS</td><td>Sony</td><td>SH800S</td><td></td></tr><tr><td>Filter tip&amp;nbsp; (1000 &amp;micro;L)</td><td>Thermo Scientific</td><td>TF112-1000-Q</td><td></td></tr><tr><td>Filter tip&amp;nbsp; (200 &amp;micro;L)</td><td>Thermo Scientific</td><td>TF140-200-Q</td><td></td></tr><tr><td>Filter tip (10 &amp;micro;L)</td><td>Thermo Scientific</td><td>TF104-10-Q</td><td></td></tr><tr><td>Filter tip (100 &amp;micro;L)</td><td>Thermo Scientific</td><td>TF113-100-Q</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Nikon</td><td>Eclipse Ni-E</td><td></td></tr><tr><td>Four-Dimensional Rotating Mixer</td><td>Kylin -Bell</td><td>BE-1100</td><td></td></tr><tr><td>Hemicellulase</td><td>sigma</td><td>9025-56-3</td><td></td></tr><tr><td>IS PCR primer</td><td></td><td></td><td>5&#039;-AAGCAGTGGTATCAACGCAGAG&lt;br/&gt; T-3&#039;</td></tr><tr><td>KAPA HiFi HotStart ReadyMix(2X)</td><td>Roche&amp;nbsp;</td><td>7958935001</td><td></td></tr><tr><td>KCl</td><td>Sinopharm Chemical Reagent Co., Ltd</td><td>7447-40-7</td><td></td></tr><tr><td>Macerozyme R10</td><td>Yakult (Japan)</td><td>9032-75-1</td><td></td></tr><tr><td>Magnetic separation stand</td><td>invitrogen</td><td>12321D</td><td></td></tr><tr><td>Mannitol</td><td>aladdin</td><td>69-65-8</td><td></td></tr><tr><td>MES</td><td>aladdin</td><td>145224948</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>yuanye</td><td>R21455-500ml</td><td></td></tr><tr><td>Microcentrifuges</td><td>Eppendorf</td><td>Centrifuge 5425</td><td></td></tr><tr><td>Micro-mini-centrifuge</td><td>Titan</td><td>Timi-10k</td><td></td></tr><tr><td>MS</td><td>Phytotech</td><td>M519</td><td></td></tr><tr><td>Nextera XT DNA Library Preparation Kit</td><td>illumina</td><td>FC-131-1024</td><td></td></tr><tr><td>oligo-dT30VN primer</td><td></td><td></td><td>5&#039;-AAGCAGTGGTATCAACGCAGAG&lt;br/&gt; TACTTTTTTTTTTTTTTTTTTTTTTT&lt;br/&gt; TTTTTTTTTTVN-3&#039;</td></tr><tr><td>PCR instrument</td><td>Thermal cycler</td><td>A24811</td><td></td></tr><tr><td>Pectolyase</td><td>Yakult (Japan)</td><td>9033-35-6</td><td></td></tr><tr><td>Plant marker lines</td><td>Nottingham Arabidopsis Stock Centre (NASC)</td><td></td><td></td></tr><tr><td>Qubit 1x dsDNA HS Assay Kit</td><td>invitrogen</td><td>Q33231</td><td></td></tr><tr><td>Qubit 2.0 fluorometer</td><td>invitrogen</td><td>Q32866</td><td></td></tr><tr><td>RNase inhibitor&amp;nbsp;</td><td>Thermo Scientific</td><td>EO0382</td><td></td></tr><tr><td>RNase-free water</td><td>invitrogen</td><td>10977023</td><td></td></tr><tr><td>Solution A</td><td></td><td></td><td>400 mM mannitol, 0.05 % BSA , 20 mM MES (pH5.7), 10 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 20 mM KCl</td></tr><tr><td>Solution B</td><td></td><td></td><td>1 % (w/v)cellulase R10, 1 % (w/v) cellulase RS, 1 %&amp;nbsp; (w/v)hemicellulase, 0.5 %&amp;nbsp; (w/v)pectolyase and 1 %&amp;nbsp; (w/v) Macerozyme R10 in a fresh aliquot of solution A</td></tr><tr><td>Sterile pestle</td><td>BIOTREAT</td><td>453463</td><td></td></tr><tr><td>Strainer (40 &amp;micro;m )</td><td>Sorfa&amp;nbsp;</td><td>251100</td><td></td></tr><tr><td>SuperScript IV reverse transcriptase (200 U/&amp;micro;L)</td><td>invitrogen</td><td>18090050</td><td></td></tr><tr><td>SuperScript IV buffer (5x)</td><td>invitrogen</td><td>18090050</td><td></td></tr><tr><td>Test tube (5 mL)</td><td>BD Falcon</td><td>352052</td><td></td></tr><tr><td>Thin-walled PCR tubes with caps (0.5 mL)</td><td>AXYGEN</td><td>PCR-05-C</td><td></td></tr><tr><td>Triton X-100</td><td>Sangon Biotech</td><td>A600198-0500</td><td></td></tr><tr><td>TSO primer</td><td></td><td></td><td>5&#039;-AAGCAGTGGTATCAACGCAGAG&lt;br/&gt; TACATrGrG+G-3&#039;</td></tr><tr><td>Vortex</td><td>Titan</td><td>VM-T2</td><td></td></tr></tbody></table>

isolation and transcriptome analysis of plant cell types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

Protoplast isolation 
This protocol is effective for the protoplast sorting of fluorescent A. thaliana root marker lines. These markers lines have been developed by the fusion of fluorescent proteins with genes expressed specifically in target cell types, or using enhancer trap lines (Figure 1). Numerous tissues and organs have been dissected into cell types expressing specific fluorescent markers in model plants and crops.
FACS population, sorted cells, and library QC 
By using a wild-type plant as a control and setting gates such as forward scatter (FSC) and side scatter (SSC), we determined a major population of cells of interest and a baseline for autofluorescence and successfully sorted the fluorescence-specific marked cells (Figure 2). The quality of the final sequencing libraries (from step 4.15) was determined by the fragment size distribution analysis. The representative results of the RNA-seq library of about 2,000 xylem-pole pericycle cells, lateral root primordia cells, endodermis/cortex cells, and lateral root cap cells are shown in Figure 3A-D.
Analysis of expression patterns 
The quality of the sequencing data can be evaluated from multiple analysis procedures, such as sequencing depth, mapping rate, and fastQC reports. The accuracy and sensitivity of the sequencing data can be demonstrated by the presence of a series of cell-type enriched genes, which can be identified from the DEG analysis between isolated cell types and the whole tissue. Genes with significantly higher expression levels in certain cell types can be identified as cell-type enriched genes. Meanwhile, the genome-browser views of each cell type can be compared side by side to show the expression levels of known marker genes and test whether the expression pattern of the marker genes can be reconstructed in the cell-type expression data. As an example, the genes that are enriched in any of the four root cell types were clustered and shown in heatmap, which showed the specificity of gene expression among different cell types (Figure 4). YUCCA3, MYB36, WOX5, and PFA1 were examined, and the expression patterns were as expected according to earlier reports21,22,23,24. Data analysis pipelines and representative raw sequencing data are available in a public repository (github.com/gaolabsjtu/root_cell_types_RNAseq).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64913/64913fig01.jpg" /> 
Figure 1: The specific cell type marker line of root and protoplast preparation. (A, left) Enhancer trap line introduction and images. (A, right) The specific cell type marker line of the root. (B) An image of the protoplast preparation before sorting. The scale bars for the lateral root founder cell, pericycle, endodermis/cortex, lateral root cap, and protoplasts represent 25 &#181;m, 100 &#181;m, 100 &#181;m, 75 &#181;m, and 20 &#181;m, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64913/64913fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64913/64913fig02.jpg" /> 
Figure 2: Establishment of FACS for specific root cell types. Example of a FACS experiment: (A) the major population using SSC and FSC; (B) the GFP-positive (+) and GFP-negative (&#8722;) gates; (C) the brightfield and (D) fluorescence images of the sorted cells. The scale bar represents 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64913/64913fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64913/64913fig03.jpg" /> 
Figure 3: Size distribution of Smart-seq2 sequencing libraries. Representative fragment size distributions of the sequencing libraries (from step 4.15) generated from xylem-pole pericycle cells (A), lateral root primordia cells (B), endodermis/cortex cells&#160;(C) and lateral root cap cells (D). (E) A small-size library with primer/adapter dimer peaks, which can still be sequenced after size selection. (F) A library with an abnormal size distribution, which indicated unsuccessful library preparation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64913/64913fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64913/64913fig04.jpg" /> 
Figure 4: Cell-type-enriched gene analysis. A DEG analysis was carried out between each cell type and the whole root; genes with more than four-fold upregulation in each cell type were identified as cell-type-enriched genes. These genes were combined, clustered, and visualized in the heatmap (upper panel) plot. The cell-type-enriched genes included many known marker genes; typical marker genes such as YUCCA3, MYB36, WOX5, and PFA1 were chosen to show in the genome browser (lower panel). Abbreviations: LRP = lateral root primordia; Endo/Cor = endodermis/cortex; LRC = lateral root cap. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64913/64913fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Volume (&#181;L)</td>
		</tr>
		<tr>
			<td>10% Triton X-100</td>
			<td>0.33</td>
		</tr>
		<tr>
			<td>RNase inhibitor</td>
			<td>0.55</td>
		</tr>
		<tr>
			<td>DTT (0.1 M)</td>
			<td>0.22</td>
		</tr>
	</tbody>
</table>
Table 1: Reaction components for the preparation of the cell lysis buffer (mixture A).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Volume (&#181;L)</td>
		</tr>
		<tr>
			<td>Oligo-dT30VN (100 &#181;M)</td>
			<td>0.44</td>
		</tr>
		<tr>
			<td>dNTPs (10 mM)</td>
			<td>4.4</td>
		</tr>
	</tbody>
</table>
Table 2: Reaction components for the preparation of mixture B.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Volume (&#181;L)</td>
		</tr>
		<tr>
			<td>SuperScript IV buffer (5x)</td>
			<td>8.8</td>
		</tr>
		<tr>
			<td>Betaine (5 M)</td>
			<td>8.8</td>
		</tr>
		<tr>
			<td>DTT (0.1 M)</td>
			<td>2.2</td>
		</tr>
		<tr>
			<td>MgCl2 (1 M)</td>
			<td>0.264</td>
		</tr>
		<tr>
			<td>TSO (100 &#181;M)</td>
			<td>0.44</td>
		</tr>
		<tr>
			<td>SuperScript IV reverse transcriptase (200 U/&#181;L)</td>
			<td>2.2</td>
		</tr>
		<tr>
			<td>RNase inhibitor</td>
			<td>1.1</td>
		</tr>
	</tbody>
</table>
Table 3: Reaction components for the preparation of the reverse transcription PCR mixture (mixture C).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cycle</td>
			<td>Temperature (&#176;C)</td>
			<td>Time</td>
		</tr>
		<tr>
			<td>1</td>
			<td>50</td>
			<td>90 min</td>
		</tr>
		<tr>
			<td rowspan="2">2-11</td>
			<td>55</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>50</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>12</td>
			<td>70</td>
			<td>15 min</td>
		</tr>
		<tr>
			<td>13</td>
			<td>4</td>
			<td>&#8734;</td>
		</tr>
	</tbody>
</table>
Table 4: Reverse transcription (RT) PCR settings for synthesizing cDNA from mRNA.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Volume (&#181;L)</td>
		</tr>
		<tr>
			<td>KAPA HiFi HotStart ReadyMix (2x)</td>
			<td>44</td>
		</tr>
		<tr>
			<td>IS PCR primer (10 &#181;M)</td>
			<td>0.88</td>
		</tr>
	</tbody>
</table>
Table 5: Reaction components for the preparation of the pre-amplification reaction mixture (mixture D).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cycle</td>
			<td>Temperature (&#176;C)</td>
			<td>Time</td>
		</tr>
		<tr>
			<td>1</td>
			<td>98</td>
			<td>5 min</td>
		</tr>
		<tr>
			<td rowspan="3">2-13</td>
			<td>98</td>
			<td>20 s</td>
		</tr>
		<tr>
			<td>67</td>
			<td>30 s</td>
		</tr>
		<tr>
			<td>72</td>
			<td>3 min</td>
		</tr>
		<tr>
			<td>14</td>
			<td>72</td>
			<td>5 min</td>
		</tr>
		<tr>
			<td>15</td>
			<td>4</td>
			<td>&#8734;</td>
		</tr>
	</tbody>
</table>
Table 6: PCR program settings for the pre-amplification reaction.

Watch this Scientific Journal Video about Isolation and Transcriptome Analysis of Plant Cell Types at JoVE.com

Isolation and Transcriptome Analysis of Plant Cell Types

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...

isolation-and-transcriptome-analysis-of-plant-cell-types

Nanyang Technological University

Research

JoVE Journal

Biology

1.3K Views.  Shanghai Jiao Tong University. The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis. 

Video: Isolation and Transcriptome Analysis of Plant Cell Types

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Genetics

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Biochemistry

Transcriptome Analysis of Single Cells

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in culture.1,2,3 In the case of the brain, regions such as the hippocampus contain a complex arrangement of different cell types, each with distinct mRNA profiles. The ability to harvest single cells allows for a more in depth investigation into the molecular differences between and within cell populations. We describe a simple and rapid method for harvesting cells for further processing. Pipettes often used in electrophysiology are utilized to isolate (using aspiration) a cell of interest and conveniently deposit it into an Eppendorf tube for further processing with any number of molecular biology techniques. Our protocol can be modified for the harvest of dendrites from cell culture or even individual cells from acute slices.
We also describe the aRNA amplification method as a major downstream application of single cell isolations. This method was developed previously by our lab as an alternative to other gene expression analysis techniques such as reverse-transcription or real-time polymerase chain reaction (PCR).4,5,6,7,8 This technique provides for linear amplification of the polyadenylated RNA beginning with only femtograms of material and resulting in microgram amounts of antisense RNA. The linearly amplified material provides a more accurate estimation than PCR exponential amplification of the relative abundance of components of the transcriptome of the isolated cell. The basic procedure consists of two rounds of amplification. Briefly, a T7 RNA polymerase promoter site is incorporated into double stranded cDNA created from the mRNA transcripts. An overnight in vitro transcription (IVT) reaction is then performed in which T7 RNA polymerase produces many antisense transcripts from the double stranded cDNA. The second round repeats this process but with some technical differences since the starting material is antisense RNA. It is standard to repeat the second round, resulting in three rounds of amplification. Often, the third round in vitro transcription reaction is performed using biotinylated nucleoside triphosphates so that the antisense RNA produced can be hybridized and detected on a microarray.7,8

In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

Neuroscience

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Isolation of High Purity Tissues from Developing Barley Seeds

Understanding the mechanisms regulating the development of cereal seeds is essential for plant breeding and increasing yield. However, the analysis of cereal seeds is challenging owing to the minute size, the liquid character of some tissues, and the tight inter-tissue connections. Here, we demonstrate a detailed protocol for dissection of the embryo, endosperm, and seed maternal tissues at early, middle, and late stages of barley seed development. The protocol is based on a manual tissue dissection using fine-pointed tools and a binocular microscope, followed by ploidy analysis-based purity control. Seed maternal tissues and embryos are diploid, while the endosperm is triploid tissue. This allows the monitoring of sample purity using flow cytometry. Additional measurements revealed the high quality of RNA isolated from such samples and their usability for high-sensitivity analysis. In conclusion, this protocol describes how to practically dissect pure tissues from developing grains of cultivated barley and potentially also other cereals.

Here we present a protocol for high purity manual isolation and quality control of embryo, endosperm and seed maternal tissues during entire barley seed development.

Understanding the mechanisms regulating the development of cereal seeds is essential for plant breeding and increasing yield. However, the analysis of ...

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

Developmental Biology

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

Isolation and Transcriptome Analysis of Plant Cell Types

Summary

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Chapters in this video

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

mRNA Interactome Capture from Plant Protoplasts

Transcriptome Analysis of Single Cells

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Isolation of High Purity Tissues from Developing Barley Seeds

Fluorescence Activated Cell Sorting of Plant Protoplasts

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses