Single Cell RNA sequencing

Single cell RNA sequencing allows you to study the transcriptome of individual cells. Different methods of single cell RNA sequencing exist, which are complementary. The method of choice will depend on your biological question and your input material:

 

 

3’ scRNA sequencing

3’ RNA sequencing allows to quantify gene expression and to identify cell populations. In 3’ scRNA-Sequencing, poly(A)+ mRNAs are reverse transcribed using a polydT primer. This polydT sequence is located directly adjacent to the barcodes required for the identification of unique cells and for sequencing. The generated cDNA molecules are amplified and then fractionated. Only the 3’ ends of the transcripts bear the barcodes, are represented in the final libraries and will be sequenced. 3’ scRNA-seq represents a relatively easy way to quantify the poly(A)+ mRNAs of single cells.

 

3’ scRNA-seq at the NGS platform

3’ scRNA-seq is available in routine at the NGS platform using the 10X Genomics pipeline. The required input material is 1,000 to 20,000 cells or nuclei and the recovery rate (yield) is around 50% of loaded cells/nuclei. The sequencing recommendations are around 50-100k reads per cell or nucleus.

Using feature barcodes, 3’ scRNA-seq can be combined with cell surface proteins expression using specific antibodies at the single cell level (CITEseq) or to increase the multiplexing capacity (Cell Hashing). Effects of CRISPR perturbations can also be assessed at single cell resolution, with direct capture of cellular sgRNAs and changes in gene expression.

 

 

 

3’ scRNA-seq at the Custom Single Cell Omics platform

The Custom Single Cell Omics (CSCO) platform currently develops an inDrop 3’ scRNA-seq method. In this approach, single cells from a cell suspension are isolated into droplets. Cells are lysed and poly(A)+ mRNAs are barcoded in the droplets. Subsequently, droplets are pooled and cDNA is further processed for sequencing.  This custom method allows to sequence a large number of cells at low costs and can be adapted to specific questions. More to come soon.

 

Single cell full length RNA sequencing

In contrast to 3’ or 5’ scRNA sequencing, single cell full length RNA sequencing covers the entire sequence of RNA molecules, allowing for example allele resolution, as well as the identification of splice isoforms and genetic variants.

 

Single cell full length transcriptomics at the Custom Single Cell Omics platform

Smart-seq3 full length scRNA sequencing is available at the Custom Single Cell Omics platform. Smart-seq3 quantifies thousands of RNA molecules per cell, displaying a striking increase in sensitivity compared to previous Smart-seq2. For more information, see Hagemann-Jensen et al, 2020 (DOI 10.1038/s41587-020-0497-0).

Starting material should count at least 500 cells per sample, and around 384 cells can currently be sequenced.

 

Single cell full length transcriptomics at the NGS platform

The development of an approach combining 10X Genomics single cell transcriptomics and PacBio Long Read sequencing techniques is underway at the NGS platform. The cDNAs generated by the 3' scRNA-seq will be used, in addition to the conventional approach, to prepare libraries compatible with the Pacific Biosciences Sequel II sequencer. This unique technology available at the NGS platform generates reads covering the entire transcript (from the 5' to the 3' end), allowing the study of isoforms with higher accuracy, without the need for assembly.

 

5’ scRNA-seq+ VDJ

5’ scRNA-seq allows to quantify gene expression and to study the immune repertoire. In 5’ scRNA-Sequencing, poly(A)+ mRNAs are reverse transcribed using a polydT primer. However, in contrast to 3’ scRNA-Sequencing, the sequencing barcodes are not adjacent to the polydT primer, but they are located at the 5' end of the transcripts. The major advantage and use of the 5′ is to combine gene expression profiling with the identification of the full V(D)J fragment (a and b chain of TCR, heavy and light chain of Igs and BCR in B-cells) in the same cells.

 

5' scRNA-seq at the NGS platform

5’ scRNA-seq is available in routine at the NGS platform using the 10X Genomics pipeline. The required input material is 1,000 to 20,000 cells or nuclei and the recovery rate (yield) is around 50% of loaded cells/nuclei. The sequencing recommendations are around 50-100k reads per cell or nucleus.

Using feature barcodes, 5’ scRNA-seq can be combined with cell surface proteins expression using specific antibodies at single cell level (CITEseq) or to increase the multiplexing capacity (Cell Hashing).