Single-cell sequencing enables the analysis of the sample at single-cell resolution. Different protocols for various applications have been established. We want to show how these differences manifest in the workflows and what that means for choosing the proper protocol for your research question.
CeGaT currently offers three single-cell-based products: 3’ Single Cell Sequencing (3’ SCR), Single-Cell Immune Profiling (SIP), and Single-Cell RNA Sequencing Flex (SCR Flex). All products are based on 10x Genomics® protocols and run on the Chromium™ X system. Within the workflows, gel beads in emulsions (GEMs) are generated on a chromium chip. All GEMs end up with many copies of one cell with the same barcode but a unique molecular identifier (UMI). So, what are the differences between the products? Let´s examine the protocols closely (see figure 1).
First, there is a difference in the sample material that can be used. Fixed cells or FFPE scrolls can be used as starting material for SCR Flex. In contrast, single cells in frozen suspension are required for 3’ SCR and SIP. Second, the protocols differ in the way the libraries are generated. For SCR Flex, the probe set is hybridized before GEMs are built. In the following steps, cells undergo RNA-templated ligation within each of these GEMs. The resulting libraries represent the protein-coding genes of the samples’ cells. In comparison, reverse transcription is performed within the GEMs for the 3’ SCR and SIP workflow. The transcription is performed either from the 3’-end or 5’-end, using poly-(dT) primers for the target amplification. Subsequently, libraries are generated representing the 3’-end of the mRNA transcript for 3’ SCR or the 5’-end of the mRNA transcripts. For SIP, the V(D)J segments are additionally amplified. All libraries can be sequenced on the Illumina platforms. The respective expression profiles of the single cells can be evaluated based on the sequenced data of all approaches.
Consequently, your sample type and research target are crucial for choosing a suitable single-cell RNA sequencing protocol. Single-Cell Immune Profiling requires fresh frozen single-cell suspension as starting material. Compared to 3’ Single-Cell RNA Sequencing, Single-Cell RNA Sequencing Flex allows you to fix the cells for later analysis. However, 3’ Single-Cell RNA Sequencing and Single-Cell RNA Sequencing Flex also differ in the library preparation and the target region. By considering these aspects, we will find the right Single-Cell RNA Sequencing product for your project. We are happy to support you!
Figure 1 | Overview of the different Single-Cell RNA Sequencing products. The differences between the products are highlighted.