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Fluidigm Corporation, a leader in mass cytometry and microfluidics technologies, today announced the introduction of a REAP-seq (RNA expression and protein sequencing) protocol for use with the C1™ system. C1 REAP-seq is a powerful multi-omic single-cell application that enables deep characterization of unique cellular subtypes and functional states by measuring the expression of both cellular proteins and RNAs. Capable of pairing with functional imaging assays that measure differences in cell size, morphology or phenotype within the clear C1 microfluidic cell chambers, C1 REAP-seq represents a significant step forward in multi-omic analysis for basic and translational research.
“C1 REAP-seq is a powerful new protocol that will enable researchers to gain greater understanding of the underlying mechanisms of cancer progression and immune response,” said Carsten Krieg, PhD, Single Cell Analytics LLC. “Providing an unprecedented view into the dynamics of protein expression at single-cell resolution, C1 REAP-seq offers a new paradigm to accelerate the identification of actionable biomarkers and drug targets.”
C1 REAP-seq was developed in collaboration with Merck for co-detection of both cellular protein and RNA using microfluidics technology. Utilizing the REAP-seq technology developed and published in Nature Biotechnology by Merck scientists (Peterson et al., 2017), C1 REAP-seq allows simultaneous analysis of up to 82 different proteins and more than 20,000 genes per cell in a single workflow. Cellular proteins are identified using third-party antibodies conjugated to unique 15-base pair barcodes that are detected by next-generation sequencing. The application is ideal for deep characterization of hundreds to thousands of cells downstream of cell atlasing studies, enabling the simultaneous detection of more cell surface protein markers than traditional cytometry approaches alone.
Schematic of REAP-seq
(a) Cells are labeled with Ab-Barcodes before compartmentalization into discrete droplets containing a bead with cell-barcode primers. Upon droplet formation the cell is lysed and the polyadenylated mRNA and AbBs hybridize to the poly(dT) cell barcoded primer. REAP-seq leverages the DNA polymerase activity of reverse transcriptase to simultaneously extend the hybridized AbB and synthesize complementary DNA from mRNA in the same reaction. The droplet emulsion is then broken and the cell barcoded AbB sequences (∼155 bp) are size fractionated from the cell barcoded cDNA derived from mRNA (>∼500 bp). mRNA and protein libraries are prepared and sequenced (see Methods). (b) Paired end sequencing reads for mRNA and protein libraries are generated on a high-throughput sequencer. The mRNA workflow is similar to previously published methods. Protein sequencing reads are first aligned using an antibody-barcode dictionary that associates each antibody with a unique 8 bp sequence Next, reads are grouped by their cell barcodes, and sequences with unique UMIs are counted for each protein and gene in each cell. The result is a digital protein and gene expression matrix where each column corresponds to a cell, and each row corresponds to a different protein or gene. Each entry in this matrix is the integer number of detected genes or proteins per cell.
“C1 REAP-seq is an important addition to our expanding menu of multi-omic single-cell applications,” said Chris Linthwaite, President and CEO of Fluidigm. “Empowering our customers with new microfluidics tools to identify meaningful DNA, RNA, protein and epigenetic biomarkers is central to powering new health care insights.”
Source – Globe Newswire