Transcriptome analysis on single cell or cell types can detect the subtle difference of gene expression being masked by heterogeneity, which is a powerful tool to uncover the elaborate intracellular regulatory mechanisms. Fluorescence activated cell sorting followed by RN-seq analysis can be performed on either single cell or bulk cell type mode with high transcript detection sensitivity and compatibility with other multi-omic approaches. First-time users of this protocol need to pay attention to some steps in the protoplast sorting and RNA-seq library preparation.
Along with Jun Zhang, Dr.Rongrong Xie, a post-doc at our laboratory, will help in demonstrating the procedure. To begin, put the Arabidopsis thaliana wild type and fluorescent marker line seeds in 20%bleach and incubate using a rotating incubator at room temperature for 15 minutes to sterilize the seeds. While working on a sterile bench, rinse the seeds three to five times in double distilled water.
Plate the wild type and reporter line seeds on half-strength MS medium with 0.8%weight per volume agar. Stratify the plants for two days at four degrees Celsius. Then grow them vertically for five days at 23 degrees Celsius under 16-hour light and 18-hour dark cycles.
Gently thaw the protoplasting solutions referred to as solution A and solution B on ice prior to the experiment. Cut off the roots from the plants using a clean blade or scissors and chop the roots into approximately 0.5 centimeter pieces. Submerge the chopped roots in 1.5 milliliters of solution B followed by gentle rotation at room temperature for 1.5 to 2 hours.
Filter the resulting root protoplasts through a 40 micron strainer mesh and rinse the mesh with one to two milliliters of solution A.Centrifuge the combined filtrates at 300 G for five minutes at four degrees Celsius. Discard the supernatant using a pipette, resuspend the cell pellet in 500 to 600 microliters of solution A, and place it on ice immediately. For cell sorting, transfer the resuspended cell solution to a new five milliliter test tube.
Fill the sheath fluid tank and check the waste tank. Then switch on the fluorescence-activated cell sorting or FACS machine. Carry out the instrument setup and auto-calibration steps on the sorter.
Select the fluorescence channels and use a wild type plant with no fluorescence as a baseline control. Adjust the sorting gate based on the fluorescence intensity and forward scatter and side scatter singlets. After adding 500 microliters of solution A into a 1.5 milliliter collection tube, start sorting and collect 2, 000 to 3, 000 cells per tube.
After sorting, immediately place the samples on ice. Centrifuge the collection tube containing the cells at 300 G and four degrees Celsius for five minutes and remove the supernatant with a pipette. Take two microliters of sorted cells and check for fluorescence using a fluorescence microscope.
Store the sorted cells at minus 80 degrees Celsius if not used immediately. For single cell index sorting, place the 96-well plate with membrane into the adapter. Calibrate the position of the plate so that the droplet falls in the center hole of the plate.
Remove the membrane from the 96-well plate and place the 96-well plate into the adapter. Select single cell sorting mode when sorting. Enter the target number of sorted cells as one and start sorting.
Before beginning the experiment, clean the bench with a surface decontaminant and 75%ethanol. Use all equipment only for sequencing library preparation. After adding one microliter of freshly prepared mixture A into the sorted sample, grind it with a sterile pestle.
Using RNAse free water, make up the volume to 14 microliters. Then transfer each sample into 0.2 milliliter thin walled PCR tube. Then prepare mixture B.Add 4.4 microliters in mixture B to each PCR tube containing samples and mix by gentle pipetting.
Incubate the samples at 72 degrees Celsius for three minutes. After incubation, immediately put the samples on ice to hybridize the oligo dT to the poly-A tail. Prepare the reverse transcription reaction mixture or mixture C in a 21.6 microliters of it to each tube containing the samples.
Subject the samples to reverse transcription reaction in a common PCR instrument per the reverse transcription program. Add 40.8 microliters of the freshly prepared pre-amplification reaction mixture or mixture D to the reverse transcription reaction product and run the pre-amplification program. The amplification cycle can be determined by quantitative PCR when the reaction just enters the exponential phase.
To purify the pre-amplification reaction products, transfer the pre-amplified products from a PCR tube to a 1.5 milliliter tube. Add 48 microliters of AMPure XP beads into each sample and gently mixed by pipetting before incubation. Place the 1.5 milliliter tubes containing the samples and beads on a magnetic separation stand for five minutes.
Carefully discard the supernatant from the samples without disturbing the beads. After resuspending them in 200 microliters of 80%ethanol, place them on the magnetic separation stand for another three minutes. After discarding the ethanol containing supernatant, air dry the samples in a tube for 10 minutes.
Resuspend the beads in 20 microliters of nuclease free water and incubate at room temperature for five minutes. Then place them on the magnetic separation stand for five minutes. Using a pipette, transfer 18 microliters of the supernatant from each tube into a new 1.5 milliliter centrifuge tube.
Finally, follow the steps described in the text to characterize the pre-library, followed by library construction, purification, and quantification. The fluorescent Arabidopsis thaliana markers lines were developed by fusing fluorescent proteins with genes expressed specifically in target cell types or by using enhancer trap lines. The fluorescence-specific marked protoplasts were successfully sorted based on the fluorescence intensity and forward scatter and side scatter singlets.
The sorted cells were visualized using brightfield and fluorescence imaging. The representative results of the RNA-sequencing library of about 2, 000 xylem pole pericycle cells, lateral root primordial cells, endodermis or cortex cells, and lateral root cap cells are shown. A small sized library with primer or adapter dimer peaks could still be sequenced after size selection.
A library with an abnormal size distribution indicated unsuccessful library preparation. The gene expression patterns were analyzed. The genes enriched in any of the four root cell types were clustered and depicted in a heat map showing the specificity of gene expressions among different cell types.
High quality and purity of target cells through correct gating and sorting and prevention of RNA degradation are key to successful library construction. For RNA-seq in the single cell mode, several methods integrated with the unique molecular identifier have been reported recently which significantly improved the performance.
