Isolation and Transcriptome Analysis of Plant Cell Types

Summary

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.

Abstract

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.

Introduction

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 tissues^1,2,3 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 studies^12,13. 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.

Protocol

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 cells (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

1. 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.
2. Rinse the seeds in double-distilled water (ddH₂O) three to five times. Perform this step on a sterile clean bench.
3. 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 °C) after stratifying for 2 days at 4 °C.

2. Protoplasting

1. Prepare the protoplasting solutions^2,5, referred to as Solution A and Solution B (see Table of Materials).
   1. 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 −20 °C for up to 1 month.
   2. 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 −20 °C for up to 1 month.
2. Gently thaw Solution A and Solution B on ice before beginning the experiment.
3. 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.
4. Filter the root protoplasts through the 40 µm strainer mesh (see Table of Materials).
5. Rinse the strainer mesh with 1-2 mL of Solution A.
6. Combine the liquids of step 2.4 and step 2.5, and centrifuge at 300 x g for 5 min at 4 °C. Discard the supernatant with a pipette, resuspend the cell pellet in 500-600 µL of Solution A, and then place it on ice immediately.
7. Transfer the resuspended cell solution to a new 5 mL test tube for cell sorting.

3. Fluorescence-Activated Cell Sorting (FACS)

1. 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).
2. Add 500 µL 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.
3. After sorting, immediately place the samples on ice, centrifuge the collection tube containing the cells at 300 x g for 5 min at 4 °C, and remove the supernatant with a pipette.
4. Take 2 µL of sorted cells, and check for fluorescence using a fluorescence microscope (see Table of Materials).
5. Store the sorted cells at −80 °C, or use them immediately for library construction (step 4).
6. 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.

4. Smart-seq2 library preparation

1. 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.
2. Prepare lysis buffer (mixture A) (Table 1) by combining 0.33 µL of 10% Triton X-100, 0.55 µL of RNase inhibitor, and 0.22 µL of 0.1 M DTT (see Table of Materials).
3. Add 1 µL of mixture A into the sorted sample, and grind with a sterile pestle. The preferable sample volume is ≤0.5 µL; use RNase-free water to make up the volume to 14 µL. Transfer each single cell sample into a 0.2 mL thin-walled PCR tube.
4. Prepare mixture B containing 0.44 µL of oligo-dT₃₀VN reverse transcription (RT) reaction primer (100 µM) and 4.4 µL of dNTPs (10 mM) (Table 2) (see Table of Materials).
5. Add 4.4 µL of mixture B to the 14 µL of sample in each tube, pipette gently to mix the sample, and incubate the sample at 72 °C for 3 min. After incubation, immediately put the samples on ice to hybridize the oligo-dT to the poly A tail.
6. Prepare the reverse transcription reaction mixture (mixture C) (Table 3) (see Table of Materials). Add 21.6 µL of mixture C to each tube containing the samples. Turn on the RT program on a common PCR instrument (Table 4).
7. Perform the preamplification reaction on ice. Prepare mixture D by combining 44 µL of 2x PCR polymerase mix and 0.88 µL of IS PCR primer (10 µM) (Table 5) (see Table of Materials). Add 40.8 µL of mixture D to the 40 µL of RT reaction product, and run the preamplification program (Table 6).
8. Purify the preamplification reaction products using Ampure XP beads (see Table of Materials). Add 48 µL of beads (0.6:1 ratio) into each sample from step 4.7, and gently mix the samples by pipetting.
9. 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.
10. Wash the beads by resuspending them in 200 µL 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.
11. Air-dry the samples for 10 min, and cover the tube to prevent contamination and cross-contamination during the air-drying.
12. Resuspend the beads in 20 µL of ddH₂O, incubate the samples at room temperature for 5 min, and then place them on the magnetic separation stand for 5 min.
13. Pipette out 18 µL of the supernatant from each tube, and transfer the samples into new 1.5 mL centrifuge tubes. Use 1 µL 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 −20 °C.
14. 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).
15. 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 >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.

5. RNA-seq data analysis

1. Trim the raw reads using Trim-Galore¹⁷ followed by mapping to the reference genome using hisat2¹⁸ (daehwankimlab.github.io/hisat2), and remove the PCR duplicated fragments using Picard¹⁹ (broadinstitute.github.io/picard).
2. 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.

Results

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 reports^21,22,23,24. Data analysis pipelines and representative raw sequencing data are available in a public repository (github.com/gaolabsjtu/root_cell_types_RNAseq).

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 µm, 100 µm, 100 µm, 75 µm, and 20 µm, respectively. Please click here to view a larger version of this figure.

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 (−) gates; (C) the brightfield and (D) fluorescence images of the sorted cells. The scale bar represents 50 µm. Please click here to view a larger version of this figure.

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 (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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Components	Volume (µL)
10% Triton X-100	0.33
RNase inhibitor	0.55
DTT (0.1 M)	0.22

Table 1: Reaction components for the preparation of the cell lysis buffer (mixture A).

Components	Volume (µL)
Oligo-dT30VN (100 µM)	0.44
dNTPs (10 mM)	4.4

Table 2: Reaction components for the preparation of mixture B.

Components	Volume (µL)
SuperScript IV buffer (5x)	8.8
Betaine (5 M)	8.8
DTT (0.1 M)	2.2
MgCl2 (1 M)	0.264
TSO (100 µM)	0.44
SuperScript IV reverse transcriptase (200 U/µL)	2.2
RNase inhibitor	1.1

Table 3: Reaction components for the preparation of the reverse transcription PCR mixture (mixture C).

Cycle	Temperature (°C)	Time
1	50	90 min
2-11	55	2 min
	50	2 min
12	70	15 min
13	4	∞

Table 4: Reverse transcription (RT) PCR settings for synthesizing cDNA from mRNA.

Components	Volume (µL)
KAPA HiFi HotStart ReadyMix (2x)	44
IS PCR primer (10 µM)	0.88

Table 5: Reaction components for the preparation of the pre-amplification reaction mixture (mixture D).

Cycle	Temperature (°C)	Time
1	98	5 min
2-13	98	20 s
	67	30 s
	72	3 min
14	72	5 min
15	4	∞

Table 6: PCR program settings for the pre-amplification reaction.

Discussion

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 input^25,26, 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 °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 well. Solution A and Solution B must be filtered using a 0.22 µm strainer. Additionally, storing Solution B at −20 °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., 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 (<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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 µm strainer	Sorfa	622110
Agar	Yeasen	70101ES76
Agilent fragment analyzer	Aglient	Aglient 5200
Agilent high-sensitivity DNA kit	Aglient	DNF-474-0500
Ampure XP beads	BECKMAN	A63881
Betaine	yuanye	S18046-100g
Bleach	Mr Muscle	FnBn83BK	20% (v/v) bleach
BSA	sigma	9048-46-8
CaCl2	yuanye	S24109-500g
Cellulase R10	Yakult (Japan)	9012-54-8
Cellulase RS	Yakult (Japan)	9012-54-8
Centrifuge tube (1.5 mL)	Eppendolf	30121589
DNase, RNase, DNA and RNA Away Surface Decontaminants	Beyotime	R0127
dNTPs (10 mM)	NEB	N0447S
DTT (0.1 M)	invitrogen	18090050
Ethanol	Sinopharm Chemical Reagent Co., Ltd	100092680
FACS	BD FACS Melody	BD-65745
FACS	Sony	SH800S
Filter tip  (1000 µL)	Thermo Scientific	TF112-1000-Q
Filter tip  (200 µL)	Thermo Scientific	TF140-200-Q
Filter tip (10 µL)	Thermo Scientific	TF104-10-Q
Filter tip (100 µL)	Thermo Scientific	TF113-100-Q
Fluorescent microscope	Nikon	Eclipse Ni-E
Four-Dimensional Rotating Mixer	Kylin -Bell	BE-1100
Hemicellulase	sigma	9025-56-3
IS PCR primer			5'-AAGCAGTGGTATCAACGCAGAG T-3'
KAPA HiFi HotStart ReadyMix(2X)	Roche	7958935001
KCl	Sinopharm Chemical Reagent Co., Ltd	7447-40-7
Macerozyme R10	Yakult (Japan)	9032-75-1
Magnetic separation stand	invitrogen	12321D
Mannitol	aladdin	69-65-8
MES	aladdin	145224948
MgCl2	yuanye	R21455-500ml
Microcentrifuges	Eppendorf	Centrifuge 5425
Micro-mini-centrifuge	Titan	Timi-10k
MS	Phytotech	M519
Nextera XT DNA Library Preparation Kit	illumina	FC-131-1024
oligo-dT30VN primer			5'-AAGCAGTGGTATCAACGCAGAG TACTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTVN-3'
PCR instrument	Thermal cycler	A24811
Pectolyase	Yakult (Japan)	9033-35-6
Plant marker lines	Nottingham Arabidopsis Stock Centre (NASC)
Qubit 1x dsDNA HS Assay Kit	invitrogen	Q33231
Qubit 2.0 fluorometer	invitrogen	Q32866
RNase inhibitor	Thermo Scientific	EO0382
RNase-free water	invitrogen	10977023
Solution A			400 mM mannitol, 0.05 % BSA , 20 mM MES (pH5.7), 10 mM CaCl2, 20 mM KCl
Solution B			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
Sterile pestle	BIOTREAT	453463
Strainer (40 µm )	Sorfa	251100
SuperScript IV reverse transcriptase (200 U/µL)	invitrogen	18090050
SuperScript IV buffer (5x)	invitrogen	18090050
Test tube (5 mL)	BD Falcon	352052
Thin-walled PCR tubes with caps (0.5 mL)	AXYGEN	PCR-05-C
Triton X-100	Sangon Biotech	A600198-0500
TSO primer			5'-AAGCAGTGGTATCAACGCAGAG TACATrGrG+G-3'
Vortex	Titan	VM-T2