High throughput transcriptomics of flow sorted cells with unique molecular identifiers
Minimizing the cost but not sensitivity of mRNA expression detection using a miniaturized CEL-Seq2 protocol.
The challenges of single cell transcript counting
Detecting mRNA expression in single-cells, for the purposes of identifying subtle transcriptome changes, requires a method that is optimized for both high sensitivity and accuracy. The CEL-Seq2 protocol of Hashimshony et al., (Cell, 2016) ticks these boxes, however costs per cell prohibit its use for high throughput applications.
The next generation protocol, developed at the Max Planck Institute of Immunobiology and Epigenetics, uses index-sorting to offer deeper cellular insights by linking the transcriptomes of single cells to their cell surface marker expression (Herman et al., Nature, 2018 & Sagar et al., Springer, 2018). This group named their mCEL-Seq2 protocol after the mosquito® nL liquid handling robot that made it feasible to significantly increase the number of cells processed per day.
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Facilitating throughput through automation
The Max Planck's mCEL-seq2 protocol is described in full here. Essentially, 384-well bar-coded primer source microplates are pre-prepared by mosquito in advance. Based on surface marker expression, individual cells are then FACS sorted into the microplates followed by cDNA synthesis reagents by mosquito.
Using this approach, one operator and one mosquito robot can process 3072 cells per day into eight x 384-well microplates ahead of pooled library preparation and sequencing.
Click here to see the miniaturized protocol graphic in detail.
The effect of protocol miniaturization on cost and sensitivity
The nanoliter to microliter pipetting range of mosquito enabled researchers to explore the effects of protocol volume reduction versus Hashimshony’s original in search of cost savings. Following extensive validation to ensure reproducibility and accuracy, it was concluded that a five-fold volume reduction provides the optimal combination of sensitivity versus cost/cell.
Already a powerful tool for the cell biologist (Herrtwich et al., Cell, 2016 & Tsai-Hsiu Lu et al., Cell Metabolism 2018) this modified mCEL-seq2 protocol is set to provide productivity gains that accelerate scientific research through a combination of deeper insights, manageable cost per cell and increased throughput.
How it all works
The automation friendly, high throughput compatible mCEL-seq2 protocol combines:
- cell barcoding to permit sample pooling ahead of library preparation
- unique molecular identifiers to reduce amplification noise
- cost effective miniaturization of protocol steps pre-pooling
to just a few hundred nanoliters using mosquito.
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