The analysis of gene products in cells is an important tool for the diagnosis of diseases and the design of new drugs in biological and medical research. At Helmholtz Zentrum München, a method of RNA sequencing (transcriptome analysis) has now been developed that pinpoints the smallest quantities of gene transcripts in individual cells. The method allows individual molecules selected in a sample to be labeled and enriched to study their cellular function. This makes it possible to selectively characterize genes in their cellular environment with high accuracy. The work has now been published in Genome Biology.

Single-cell RNA sequencing relies on the study of molecular transcripts created by active regions of the genome in individual cells. Depending on the type and stage of development, cells activate different genes, which are read by RNA molecules and translated into proteins. The number of mRNA molecules or transcripts per gene in a given cell can inform us about their identity and their physiological response to internal or external signals. These can be diseases, aging processes, environmental influences or reactions to pharmacologically active substances. However, the detection of genes that are only expressed in moderate to low concentrations poses major challenges to currently used single-cell RNA sequencing techniques. The analysis primarily identifies so-called housekeeping genes, which, in contrast to dynamically regulated genes, are constantly expressed.

This problem bypasses that of a team around Dr. Micha Drukker, Institute of Stem Cell Research, and PhD student Fatma Uzbas developed the BART-Seq method by enriching selected transcripts for sequencing. BART-Seq stands for "Barcode Assembly for Targeted Sequencing". Primer sets and DNA barcodes are combined so that they can amplify the transcripts of desired genes. "We have developed a novel technique for indexing primers with DNA barcodes through a simple synthesis reaction," explains Micha Drukker. Sequencing transcripts can be traced back to the individual cells from which they are derived. At the same time, since the analysis targets only the selected genes, it is possible to obtain high-resolution information about these genes and thus to characterize each cell individually.

The process is inexpensive and does not require specialized and expensive instrumentation. Any research group with access to next-generation sequencing instruments can use BART-Seq for both single-cell and aggregate analysis of RNA or genomic DNA from thousands of samples.

Together with Philipp Angerer, Nikola Müller and Fabian Theis from the Institute for Computational Biology of Helmholtz Zentrum München, Drukkers has developed software for the design of primers and barcodes as well as for the analysis of sequencing data. To make the method accessible to all research groups, the software is freely available on the internet.

Micha Drukker and his teammates want their process to become an integral part of the Basic and Application Research Toolkit. Especially drug screening projects, such as measuring the response of cultured β-cells to drugs or, for example, targeting genome sequences with CRISPR-Cas9 could benefit greatly from BART-Seq.