Creation of the Single Cell platform at the IRS-UN co-financed by the SIRIC ILIAD
The platform is located on the 6th floor of the Health Research Institute of the University of Nantes (IRS-UN). It is managed by a molecular biology technician from the CRCINA, trained in single cell technologies implemented in the Chromium equipment (10X Genomics). For the SIRIC ILIAD, the IRS-UN single cell platform is used to perform single cell transcriptome (scRNA-seq) of samples from patients with multiple myeloma and breast cancer included in the MYRACLE, THEREX, and EPICURE prospective cohorts. The scRNA-seq of tumour cells and their environment is one of the tasks to be carried out within axes 6, 7 and 8 of the SIRIC.
Principle of the technique
scRNA-seq remained limited until the publication of drop-seq technology (Macosko et al. Cell 2015) and the commercialisation of turnkey solutions such as Chromium, making the whole process relatively easy and fast. For example, Chromium can produce 80,000 transcriptomes in just 2 days with a single technician.
The principle of drop-seq is as follows: using a plastic chip, the technician places live cells in a first well, oil in a second well and capture beads / RT lysis buffer in a third well. The chip is then inserted into the machine, the contents of the 3 wells pass through microchannels and collect in a chamber where the cells and capture beads are encapsulated in an aqueous droplet surrounded by oil. At the end of the cycle (10 min), the droplets are collected in the fourth well. When a cell is encapsulated in the droplet, it lyses and immediately releases its RNA. The polyA RNAs are then captured by the polyT probes bound to the capture bead. The capture efficiency is estimated at 30%. A cell barcode and an IMU are located upstream of the polyT probe sequence. After droplet collection, the captured polyA RNAs are retrotranscribed to a cDNA incorporating the barcodes. The single-stranded cDNAs bound to the capture beads are then processed to form the scRNA-seq library for pair-end sequencing (25-50,000 pair-end reads per cell). Read 1 contains the cell and IMU barcodes while read 2 contains the cDNA sequence (3’UTR of the captured polyA RNA). The Cell Ranger software (10x Genomics) processes the raw data and generates an expression matrix containing the number of unique IMUs for each gene in each cell.
The IRS-UN’s single cell genomics platform works closely with
- The cytometry and cell sorting platform (CytoCell) located on the same floor, for cell preparations upstream of Chromium
- The genomics and bioinformatics platform (GenoBiRD) located in the same building, which integrates very high-throughput sequencing (purchase of a latest-generation Illumina NovaSeq 6000 sequencer in the first quarter of 2019) and bioinformatics analyses supported by a dedicated computing and storage infrastructure (1,456 cores, 5.5 TB of RAM, 600 TB of storage), directly connected to the sequencers.