CEL-Seq2 – sensitive highly-multiplexed single-cell RNA-Seq

Single-cell transcriptomics is a transformative method with tremendous potential to illuminate the complexities of gene regulation. Single-cell transcriptomics requires a method that is sensitive, accurate, and reproducible. The CEL-Seq method is the first method to use in vitro transcription (IVT) for the amplification, thereby eliminating the requirement for a template-switch step which is thought to reduce efficiency. The method uses early barcoding, enabling highly-multiplexed analysis, and 3′ end tagging enabling accurate estimation of expression levels without having to account for gene length and with fewer sequencing reads required

Now, a team led by researchers at the Israel Institute of Technology have developed CEL-Seq2, a modified version of the CEL-Seq method, with threefold higher sensitivity, lower costs, and less hands-on time. The team implemented CEL-Seq2 on Fluidigm’s C1 system, providing its first single-cell, on-chip barcoding method, and detected gene expression changes accompanying the progression through the cell cycle in mouse fibroblast cells. The researchers also compare with Smart-Seq to demonstrate CEL-Seq2’s increased sensitivity relative to other available methods. Collectively, the improvements make CEL-Seq2 uniquely suited to single-cell RNA-Seq analysis in terms of economics, resolution, and ease of use.

Changes introduced to the protocol

a An outline of the CEL-Seq2 method is shown with the steps modified from the original CEL-Seq indicated in red. b Distribution of the reads in two libraries prepared with or without ligation from the same amplified RNA of ten replicates of 100 pg clean RNA. c, d Number of transcripts (c) and genes (d) detected. Error bars indicate standard error

Availability – The analysis pipeline is available at https://github.com/yanailab/CEL-Seq-pipeline.