The Importance of Pilot Studies for scRNA-seq

Setting Up for Success: Exploring Pilot Studies for scRNA-seq

Cell Number and Cell Quality with PIPseq

PIPseq is a flexible, scalable and instrument-free single-cell RNA sequencing (scRNAseq) method based on rapid templated emulsification of cells and barcoded hydrogel templates without microfluidics. In contrast to microfluidic emulsification, in which droplets are created sequentially and thus their number scales with instrument run time, templated emulsification generates droplets in parallel by bulk self-assembly, and, thus, the number of droplets (and cells that can be barcoded) scales only with container volume. Two important factors for producing high-quality data using this gentle, instrument-free microdroplet technology are the cell numbers used per experiment and the quality of the cells. 

PIPseq is optimized for efficient and sensitive transcript capture from live cells, and therefore, quick isolation and mild dissociation of cell types is essential, and should be confirmed in a pilot experiment before committing to the actual experiment. Completion of a pilot study prior to scale up is especially important when working with any new sample types.

The Benefits of a Pilot Experiment

Purchasing a T2 kit ($300/sample) along with the T20 or T100 kits planned for the actual experiment is an economical way to complete an inexpensive pilot study prior to committing to large-scale data production. A pilot study is key to an informed experimental design for generating the type of sequencing results that you are looking for. There are many processes to evaluate during a single cell sequencing project. For some projects, the sample preparation process might be complex. Steps such as dissociation, enrichment, labeling cells with antibodies, accurate quantitation of cells, determining cell viability, the use of proper pipetting force and centrifuge speeds, and evaluation of third-party methods to remove debris and dead cells should all be evaluated prior to scale up. These steps can vary for each sample type.

The T2 kits have the same chemistry as the higher cell capture kits, so you can assess each step of scRNAseq process and complete any optimization needed, prior to scaling up with larger reactions that may require a significantly higher read depth and a more expensive sequencing run: 

• T2: 100M reads per sample 
• T20: 800M reads per sample 
• T100: 4B reads per sample 

*Based on Fluent BioSciences 20,000 reads per cell recommendation. 

If unsure about sample type heterogeneity, it might be necessary to run the pilot experiment with T20 reactions to identify the rarity of the target subpopulations. This will also enable approximation of the cell capture rate percentage from the input cells for the target sample type, as this can vary. This type of information is valuable to have prior to running large experiments requiring 100K – 1M captured cells per sample. 

Assessment of mRNA Quality

An assessment of the mRNA quality from the sample is necessary to determine if the new sample type and associated upstream processes are sufficiently optimized. This is one of the more common failure modes for scRNA-seq and this can be evaluated in a quick pilot study without even taking the T2 reactions through to sequencing. Running through the PIPseq workflow and completing two important quality check steps can provide a lot of useful information. The first step checks the cDNA quality/yield and verifies the mRNA has not been critically degraded. The final step ensures the NGS libraries are sufficient for Illumina sequencing. Passing both quality check steps is essential for producing quality sequencing results.

If the cDNA or library yields are low, it might be necessary to increase the number of cycles used for whole transcriptome amplification or Index PCR amplification for that sample type. If there is no yield, it could be an indication that an error occurred during the PIPseq workflow that can be worked out prior to scale up. If critically degraded mRNA is observed (cDNA average fragment size < 500 bp) during the pilot study, the resulting sequence quality will be poor and is likely to contain many empty adapter reads and poly A sequences. 

When working with a new sample type for the first time, the application scientists at Fluent Biosciences recommend adding RNase inhibitor during the cell capture step of the PIPseq workflow to substantially reduce the risk of any mRNA degradation being caused by the PIPseq workflow itself. That way if any mRNA degradation is observed, it is likely due to upstream processes. The researcher can then focus efforts on improving cell viability, reducing sample preparation time, evaluating more gentle dissociation or enrichment processes, or testing third-party methods for further removal of dead cells and debris. Further information on this topic can be found in our blog post available here: https://www.fluentbio.com/blog/common-mistakes-in-cell-prep-for-single-cell-sequencing/

Assessment of Sequencing Metrics

If you take your pilot experiment through sequencing, this will provide even more information to help guide your experiment design. To assess sample quality, it is informative to review the rank plot and clustering results. You can also review key sequencing metrics to verify the data quality is good prior to deeper sequencing with larger cell numbers. These metrics include the percentage of mapped reads, captured cell count, percentage of reads in cells, percentage of reads in mitochondria, duplication rate, genes/cell, and transcripts/cell. Further information on this topic can be found in our blog post available here:  https://www.fluentbio.com/blog/data-analysis-with-pipseeker-software/

When researchers are comparing wild type vs. treated or mutant conditions, the cDNA in these treated groups may have lower quality mRNA that is an inherent part of their mutant/treatment system. Discovering this during a pilot study allows for a more informed experiment. It might be necessary to prepare two or more PIPseq reactions from the same single cell suspension to reach the desired cell number for the experiment or to reach an equivalent cell capture rate as the wild-type sample. After the technical replicates are sequenced (ideally in the same sequencing run to limit batch effects), the filtered matrices output from the PIPseq analysis for the replicates can be merged together so they can be examined as a single sample. 

The scalability, cost and efficiency of a single-cell experiment can vary significantly across different single cell sequencing technologies. With PIPseq, the scalability is not limited by expensive microfluidics or micro-titer plate requirements, so scale-up is a breeze once you have verified the sample preparation processes have been properly optimized and the input number of cells or nuclei planned for the experiment are adequate based on the information gained from your pilot study.

Get Started With The Most Cost Effective Pilot Study Today:

We are currently offering new customers that purchase one or more T20 or T100 kits the opportunity to purchase a T2 kit at a 50% discount ($150 per sample) to use for their pilot studies. Contact sales@fluentbio.com for more information.

PIPseq Best Practices
Adequate Cell Number: Both sample heterogeneity and subpopulation frequency should be taken into consideration to define adequate cell numbers required for an experiment. The use of a T2 kit is appropriate for homogenous or enriched samples (e.g., subpopulations ≥ 20% of all cells). If you are looking for rare subpopulations (e.g., <5% of all cells) without using prior enrichment steps which can be damaging to cell health, then using the Fluent BioSciences T20 kit is recommended. When trying to identify rarer subpopulations (e.g., <1% of all cells), then a T100 kit is more appropriate, as the additional cells captured would make the rare cell population more likely to cluster separately from the other cel
Accurate Counting: It is important to measure the input cell concentration using viable cells from the final resuspension with an accurate and reproducible quantification method, such as acridine orange/propidium iodide (AO/PI) staining with an automated fluorescent cell counter or trypan blue staining with a hemocytometer. For some cell types, cell viability may be negatively affected when cells are kept in suspension for a prolonged period of time, so cell suspensions should be loaded into the PIPseq assay as soon as possible after preparation.
Adequate Quality: Sample quality is critical for optimal results. Ideal cell suspensions are 90% viable and contain minimal debris or aggregates prior to diluting with the PIPseq cell suspension buffer.The best option is to start from fresh cells or tissue whenever possible. We recommend nuclei sequencing when starting from frozen tissue. It is important to work quickly and minimize unnecessary handling steps. If cells are handled too roughly, cells will lyse, releasing background mRNA. To minimize damage during sample preparation, pipetting and centrifugation should be kept to a minimum. A tightly packed cell pellet requires extra pipetting, which can damage cells from shearing effects. Pipetting steps should be slow and gentle. The use of wide-bore pipette tips is recommended to minimize the shear force on the cell suspension during certain points in the cell preparation protocol, even when working with small cell types. It might be necessary to use narrow bore pipette tips in some cell types to reduce clumps. When optimizing the workflow, pay close attention to using the minimum speed and force necessary for a given cell type, as this will vary.
Increase Cell Quality Using Third-Party Methods: Fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) can be beneficial for the enrichment of target cell populations and can facilitate the exclusion of dead or damaged cells. Enrichment steps also add a lot of time and can stress the cells causing an overall decrease in cell health. It might be necessary to include a third-party method to increase cell viability post enrichment or after tissue dissociation. These methods include Miltenyi Biotec’s Debris Removal Solution, which uses a fairly gentle process to remove dead cells and debris, or the Dead Cell Removal Kit, which effectively removes dead cells but requires a higher cell count. An appropriately sized tip strainer or filters may be used to remove debris and dead cells, as dead cells aggregate and form clumps. It is important to note that each of these methods will result in some cell loss.