making sense of single cell genomics with The C1

What I absolutely love about my career in sales, is that I am always able to learn something new and exciting from brilliant minds.

Over the past 3 years, the C1 single cell auto prep system (C1) has fueled many single cell projects, and many single cell discoveries. In my role I get to speak with many amazing researchers regularly, and am constantly amazed by the projects that are being done. I’m equally lucky to be working with a team of amazing Application Scientists, who help me make sense of it all.    

Single cell biology requires many considerations vs bulk cell studies. Here are a couple of topics that might help in understanding why the approach Fluidigm has taken helps forward your single cell projects faster: 

“How many cells do I need to look at? 96, 800, 10,000???” This question can be paralyzing to many projects.  There isn’t a single algorithm that is the magic bullet to answering the exact number of cells to run. BUT, there are other things to consider that can take your project off the ground faster:

What you do need to consider is taking an approach that:

1. allows you to minimize your costs, while progressing your research
2. gives you the most flexibility
3. allows you to QC your workflow in a very manageable way
    

1. allows you to minimize your costs, while progressing your research

with The C1 you can capture up to 96 cells on our standard IFC or 800 cells on our High-Throughput IFC

• that means, for single cell mRNAseq experiments, you can run a single Illumina HiSeq lane with cDNA from up to 4 standard IFCs (max 384 cells) or a run of the High-Throughput IFC on a single HiSeq Lane

• Assumptions: $2000/lane, Illumina HiSeq @ 250M Reads / Lane

 Generally speaking: if you have an extremely heterogeneous population or are after a rare cell type or rare transcript, you need to look at more cells. But it’s hard to tell in the beginning what size your project will need to be, and how many cells you will have to go through. That is something that you learn as your project progresses. That’s the advantage of The C1. You can scale your projects based on data from runs, and other indications as your project is moving along.    

Other approaches might allow you to isolate 8,000, 10,000 or even 40,000 cells, but then you are forced to consider sequencing that many cells, which can be cost prohibitive up front. How many lanes would that be to run 10,000 – 40,000 cells? With The Fluidigm C1 single cell auto prep system, you can go in a step wise fashion that is cost conscious, and manageable from a sample throughput perspective. Also, you may be able to recapitulate the bulk with modest cell numbers. Consider: 

• Kim et al (2015) “Single-cell mRNA sequencing identifies subclonal heterogeneity in anti-cancer drug responses of lung adenocarcinoma cells” indicated that 25-35 LUAD cells was all that was needed to largely recapitulate the transcriptional diversity seen in bulk cells
• Shalek et al (2013) “Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells” ran 18 single cells from bone marrow derived dendritic cells and three pools of bulk DCs (composed of 1000 cells each)
• Treutlein et al 2014 “Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq” measured 198 individual cells at four different stages of alveolar differentiation. They initially “sequenced transcriptomes of 80 individual live cells of the developing mouse lung epithelium” and found concordance
• Burns et al (2015) “Single-cell RNA-Seq resolves cellular complexity in sensory organs from the neonatal inner ear” PCA analysis  and unbiased clustering (158 utricle  cells and top 195 genes) identified 7 clusters of the cells that were assigned to 3 primary cell populations (TEC, SC and HC)
• Many more examples of key discoveries being made with less than 1000 cells. See attached Bibliography. 
  
  2. Gives you the most flexibility

many single cell techniques or approaches allow you to do only  3’ end counting on single cells. But what if you also or instead want to look at  full length cDNA or DNA from single cells. The C1 is designed to allow you to do that: Applications supported on the C1:

• Full length cDNA for mRNAseq
• 3’ end counting on our 800 HT IFCs
• Whole Genome amplified DNA from single cells: from there you can sequence the cell’s Whole genome, its whole exome or perform targeted resequencing of regions within that cell’s genome
• Targeted cDNA amplification for fownstream qPCR of just those cDNAs (Ranging from 1 target up to nearly 800 miRNAs)
• New protocols:
  • ATAC seq – uncover the epigenetic states of single cells, including chromatin structure and DNA methylation
  • C1 Cage – use random priming, UMIs and paired-end sequencing to allow molecular counting of mRNAs, long non-coding RNAs, incl. non-poly A transcripts
  • SMART seq2 –  Full length cDNA for mRNA-seq with the popular homebrew template switch chemistry using a locked nucleic acid template switch primer.
  • SMRT-Seq v4 – Updated Clontech v4 chemistry for improve, more sensitive, less expensive full length cDNA for mRNAseq
  • STRT seq – 5’ end counting sequencing chemistry including UMIs
  • More releases every month…

Side note for flexibility: Single cell qPCR is a POWERFUL alternative to single cell mRNAseq: It’s a lot faster and a lot less expensive, plus it has the advantage of explicit hypothesis testing, meaning there is no sifting through sequencing data to “find something interesting”.

Sometimes it can feel like the only option you have is to do single cell mRNAseq. That isn’t true. Think about single cell qPCR with the C1 + Biomark HD. It is faster than sequencing, a lot less expensive, and with the ability to look at 96 genes per cell within two days…yes 96 genes per 96 cells in 2 DAYS days max.

        3. allows you to QC your workflow in a very manageable way 

The C1 makes it easy to do a quick AND CRITICAL visualization check whether: (A) you have a cell or not, (B) if it is alive or dead (if you stained for live/dead) (C) if you have a single cell or multiple, (D) assess the phenotype for each captured cell, be it the GFP reporter construct or some other cell surface tag.

This quick and simple quality check, that can ensure that you move forward with confidence, and not waste any reagents or consumables on dead cells or non-target cell types that might not yield any usable cDNA or DNA; The C1 will give you all the power to downstream process only the best cells or the right cells for your experiment VS sequencing everything on limited QC of your cells. All you need is a regular bench microscope with fluorescent capabilities if you would like to visualize your stain. 

So in summary, the C1 allows you to avoid the short comings of techniques that solely focus on looking at thousands of cells on a single end-counting sequencing chemistry. The C1 helps in taking a manageable, cost effective, flexible and reassuring path towards amazing single cell discoveries. 

Please let me know if you have any questions or comments. I look forward to learning from your experiences, hearing about your projects and am eager to share my experiences and knowledge with you. 

Cheers,
Zeeshan