cellenONE is highly versatile as it is suitable for most library preparation protocols in a variety of consumables formats (96, 384, 1536 wp and nanowell substrates like cellenCHIP™).
Unlike other single cell technologies, cellenONE has demonstrated outstanding cell recovery (up to 95%), which also position it as an ideal tool for rare cells isolation.
A 3’ scRNA-Seq protocol was developed in cellenCHIP using a range of commercially available reagents.
The following steps describe the workflow using cellenCHIP and cellenONE®
3 different reverse transcriptase were compared (RTase_1 to _3).
To validate these protocols, two cell types (HEK and NIH_3T3) were sequentially isolated as a checkerboard pattern together with several controls.
OligodT barcoded wells were filled with 100nL of a buffer mix containing (lysis and RT reagents).
After single cells isolation, cellenCHIP were sealed and placed into a thermocycler for reverse transcription and template switching reactions.
cellenFUNNELS were used for pooling reaction mixture into microcentrifuge tubes and remaining steps (cDNA amplification, tagmentation and library amplification) were carried out as described in the literature.
After sequencing, and bioinformatic analyses all 3 enzymes showed reproducible results.
RTase_1 performed best with up to 7000 detected genes per single cell with a median of 5000 detected genes per single cell.
All libraries were analysed (Bioanalyzer) and consistently displayed fragments of about 500bp.
Compatible with a range of commercially available enzymes and reagents.
Method suited for various samples size from few (<100) to 1000’s cells.
Simplified protocol with combined lysis and RT in 100nL in cellenCHIP™.
Low number of PCR cycles (<18) to minimize data bias, and limit UMI replicates.
High reproducibility observed within biological replicates.
High quality with minimal to null background.
High performance with up to 7000 genes detected per single cell (median 5000 genes detected) with a low quantity of reads needed (median 100,000 mapped reads per single cell).
In collaboration with the Department of Neurology at Weill Institute in San Francisco, a study performed aiming to isolate single immune cells in multiple sclerosis patient´s cerebrospinal fluid (CSF) samples for subsequent single cell RNA sequencing.
The main challenge associated with such sample is the very low number of cells present in these samples which can vary from only 20 to 500 cells per 20mL of patient’s CSF.
On average, recovery reached 95%, meaning most of the cells present in each sample were successfully isolated as single cells (number of single cells successfully isolated with cellenONE X1 over average cell number for each sample).