High throughput transcriptomics of flow sorted cells with unique molecular identifiers
Minimizing the cost but not the sensitivity of single cell RNA-seq using a miniaturized CEL-Seq2 protocol.
A novel CEL-Seq2 protocol for higher-throughput scRNA-seq
Combining index-sorting with single cell RNA-seq offers the possibility to pre-select subpopulations of interest, and to link transcriptome data with cell surface marker expression. A reliable scRNA-seq workflow requires a cDNA synthesis method that is optimized for both high sensitivity and accuracy. The CEL-Seq2 protocol of Hashimshony et al. (Genome Biol. 2016) ticks these boxes, however reagent cost becomes a limiting factor when scaling up the numbers of cells to be analysed.
The next generation CEL-Seq2 protocol, developed at the Max Planck Institute of Immunobiology and Epigenetics, relies on automated nanolitre-scale liquid handling to achieve a step-change in throughput (Herman et al., Nature Methods 2018 & Sagar et al., Methods in Molecular Biology, 2018). This group named their mCEL-Seq2 protocol after the mosquito® LV genomics nL liquid handling robot that made it feasible to process thousands of cells per day.
Continue reading to learn more, or click here to watch the full webinar presentation from a senior scientist at the Max Planck Institute discussing mCEL-Seq2.
Lab automation facilitates processing of FACS-sorted single cells
The new mCEL-Seq2 protocol is described in full here. Essentially, 384-well microplates are pre-prepared with lysis buffer using the mosquito liquid handler. Based on surface marker expression, individual cells are then sorted into the plates, followed by the addition of cDNA synthesis reagents with mosquito.
With this approach, one operator can process eight 384-well plates or 3072 cells per day. Subsequent in vitro-transcription and library preparation are carried out in bulk.
Click here for an enlarged version of the workflow graphic.
mCEL-Seq2 is a cost-effective and highly sensitive scRNA-seq method
The nanolitre pipetting range of mosquito allows up to tenfold volume reduction vs. the original CEL-Seq2 protocol. Sensitivity and accuracy of miniaturized mCEL-Seq2 were extensively benchmarked against the full-volume protocol (the figure on the right is an excerpt from (Herman et al., Nature Methods 2018).
Following extensive validation, it was concluded that a five-fold volume reduction provides the optimal combination of sensitivity versus cost.
Low-volume cDNA synthesis from single cells
The routine mCEL-seq2 process relies on cost-effective miniaturization of protocol steps pre-pooling to just a few hundred nanoliters using mosquito (figure, left).
This protocol combines:
- index sorting for simultaneous measurement of cell-surface marker expression and single-cell transcriptomes
- unique molecular identifiers to reduce amplification noise
- cell barcoding to permit sample pooling ahead of library preparation
Please contact us for further information from our Genomics specialists.
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