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5 tips to make your single-cell RNA-seq experiments a success
Making RNA-seq libraries from the very low mass of RNA in single cells can be tricky. These tips will help you avoid common problems so you can get the most out of your single-cell RNA-seq experiments!
1. Do a pilot experiment before processing your samples
Even those familiar with single-cell RNA-seq workflows may need to optimize experiments when they use different sample types. Pilot experiments can help identify any issues early and avoid wasting reagents and time on larger experiments. Pilot experiments typically include a few experimental samples, positive controls, and negative controls. The performance of the control reactions can help you identify issues with your experimental technique (discussed in tip 2). When your controls perform as expected, the cDNA yield and size distribution can inform changes to your experiment to get the best performance from your experimental samples. As different cell types can vary widely in their RNA content, you may need to adjust the number of PCR cycles to obtain optimum yield for downstream library preparation.
|Sample type||Approximate RNA content (mass per cell)|
|Jurkat cells||5 pg|
|HeLa cells||5 pg|
|K562 cells||10 pg|
|2-cell embryos||500 pg|
Table I. RNA mass per cell for commonly used sample types.
2. Always do positive and negative control reactions
Whether you've done single-cell RNA-seq one or one thousand times before, these control reactions are invaluable for troubleshooting your experiments. All of Takara Bio's NGS kits include positive control samples, and the best positive control has an RNA input mass similar to your experimental samples (e.g., 10 pg of RNA for as a starting point for single cells, or see the table above). Similarly, the best negative control is one treated the same as your actual samples (e.g., mock FACS sample buffer). Some new users may see low cDNA yields in their positive control samples. Until you are comfortable with the protocol, you may want to test two positive control inputs (e.g., 10 pg and 100 pg). New users can sometimes also see a high background in their negative controls, which is a critical issue (see tips 4 and 5 for ways to decrease background).
3. Ensure that your cells are suspended in, and/or FACS-sorted into, an appropriate buffer
Reverse transcription (RT) conditions in single-cell RNA-seq experiments are carefully calibrated to ensure maximum yield from a very low amount of starting material. The presence of media, DEPC, RNases, magnesium, calcium, or EDTA that are carried over with cells can interfere with the RT reaction and reduce cDNA yield and sensitivity. If possible, we recommend washing and resuspending your bulk cell suspension in EDTA-, Mg2+- and Ca2+-free 1x PBS, especially if using enzymatic dissociation techniques such as trypsinization. If you are using FACS to isolate single cells, you can also resuspend your cells in a buffer like BD FACS Pre-Sort Buffer, which can help maintain cells in suspension and is also EDTA-, Mg2+- and Ca2+-free. If possible, we recommend using EDTA-, Mg2+- and Ca2+- free 1x PBS as a sheath fluid, but other sheath fluids that contain low concentrations of EDTA are unlikely to impact the RT reaction.
Different protocols have different recommendations as to the appropriate buffer and volume to FACS-sort your cells into, but a good rule of thumb is to sort into freshly-prepared lysis buffer containing an RNase inhibitor. See the table below for recommendations tailored to our single-cell RNA-seq kits. If your experiment requires you to adjust one or more of these parameters, a pilot experiment is the best way to determine the impact of these deviation(s). For example, compare the cDNA yield and size distribution of cDNA made from 10 pg of control RNA with and without the amount of media or buffer that you expect to come along with your cells.
|Kit||Recommended FACS collection buffer||Volume||Contains||Alternative collection buffers|
|SMART-Seq v4||1X Reaction Buffer||11.5 µl||Lysis buffer and RNase inhibitor||<5 µl Mg2+- and Ca2+-free 1X PBS|
|SMART-Seq HT||CDS Sorting Solution||12.5 µl||Lysis buffer, RNase inhibitor, and CDS primer||11.5 µl Plain Sorting Solution (without CDS oligo) or <5 µl Mg2+- and Ca2+-free 1X PBS|
|SMART-Seq Stranded||Mg2+- and Ca2+-free 1X PBS||7 µl||Phosphate-buffered saline||8 µl 1.25X Lysis Buffer Mix|
Table II. Recommended and alternate FACS collection parameters for SMART-Seq v4, SMART-Seq HT, and SMART-Seq Stranded kits. Exact buffer recipes for our recommended FACS collection buffers are included in the relevant user manuals. We note that sorting into an alternative buffer may impact the performance of our kits and will require changes in the suggested RT reaction master mixes.
4. Work quickly
Once cells have been deposited into the wells of plates or tube strips and gently centrifuged at 100g, samples should either be processed immediately or snap-frozen in dry ice and stored at –80°C until processing. Minimizing the time between cell collection, snap-freezing, and cDNA synthesis steps will reduce RNA degradation and unwanted changes in the transcriptome profile. Throughout the protocol, reducing handling time limits the chance of sample contamination or degradation.
5. Practice good RNA-seq lab techniques
Working with ultra-low-input samples is difficult, and it is easy to contaminate or lose sample material over the course of an experiment. Always wear a clean lab coat, sleeve covers, and gloves throughout the procedure. Remember to change gloves between steps in the protocol. It is also a good practice to maintain separate pre- and post-PCR workspaces. Ideally, the pre-PCR workstation should be in a clean room with positive air flow, which will greatly decrease the risk of amplicon or environmental contamination. Using RNase- and DNase-free and low RNA- and DNA-binding plasticware (pipette tips, plates/tube strips, etc.) should decrease sample loss. Bead cleanup steps can be another significant cause of sample loss. Make sure to allow the beads to fully separate before removing the supernatant or you will lose a lot of material. Using a strong magnetic device will improve and speed this separation. After ethanol washes, be sure to follow recommendations in the protocol for drying and hydration times.
Takara Bio is an expert in the field of single-cell RNA-seq, and we have pioneered and systematically advanced this technology for years. We offer a range of single-cell RNA-seq kits, including oligo-dT and random priming solutions. These tips are intended to minimize your single-cell RNA-seq difficulties, but if you have specific questions about our kits or are running into experimental issues, our technical support team is standing by and happy to help.
Additional reading for single-cell RNA-seq experiments
Haque, A., Engel, J., Teichmann, S. A. & Lönnberg, T. A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications. Genome Med. 9, 75 (2017).
Kolodziejczyk, A. A., Kim, J. K., Svensson, V., Marioni, J. C. & Teichmann, S. A. The Technology and Biology of Single-Cell RNA Sequencing. Mol. Cell 58, 610–620 (2015).
Wu, A. R., Wang, J., Streets, A. M. & Huang, Y. Single-Cell Transcriptional Analysis. Annu. Rev. Anal. Chem. 10, 439–462 (2017).
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