Illumina Single Cell 3' RNA Prep technology is optimized for efficient and sensitive transcript capture from live cells, and therefore, quick isolation and mild dissociation of cell types is essential and should be confirmed in a pilot experiment before committing to the actual experiment. It is also helpful to complete a pilot prior to scaling up, increasing sample number, or using Supplemental Enrichment and Amplification (SEA) kit applications. Completion of a pilot study before scaling up is especially important when working with any new sample types. Pilot studies are also valuable for labs new to single cell sequencing.
General Pilot Study Recommendations
The T2 kits have the same chemistry as the higher cell capture kits, so T2 kits can be used to assess each step of the scRNAseq process and complete any optimization needed, before scaling up to larger reactions that may require a significantly higher read depth and higher output sequencing runs. The following list of recommendations can help with preparation for an effective pilot study before scaling up, when working with a new sample type, or for users new to single cell.
Purchasing a T2 kit along with the T10, T20, or T100 kits is an economical way to complete a pilot study.
Processing a small number of samples (1-4 samples) as opposed to starting with large studies with many treatments or conditions.
Optimizing steps of cell or nuclei preparation for a new sample type including:
Dissociation
Accurate quantitation of cell/nuclei counts
Cell viability assessment in Illumina Single Cell - Cell Suspension Buffer
Proper pipetting force
Centrifugation speeds
Labeling cells
Enrichment
Use of third party kits to remove debris and/or dead cells
Comparing wild type (WT) versus treated or mutant conditions
Treated/mutant groups may have lower quality mRNA that is inherent to their biology.
Discovering this during a small pilot prior to a large-scale study allows for a more informed experiment.
It may be necessary to prepare 2+ Illumina Single Cell reactions for the treated/mutant group to achieve the same number of captured cells as the WT.
Technical replicates of treated/mutant groups should be sequenced in the same sequencing run and merged using analysis software to be analyzed as a single sample.
Completing Illumina Single Cell T2 reactions through cDNA QC and Library QC successfully without sending for sequencing can be economical.
If taken through sequencing, this will provide even more information to help guide the larger experiment design when scaling up.
Review key sequencing metrics to verify data quality is good before deeper sequencing with larger cell numbers including: - Captured cell count - % reads mapped - % reads in cells - % mitochondrial reads - Duplication rate - Median Genes/cell - Median Transcripts/cell
Alternatively, after completing all sample preparation processes upstream of the Illumina Single Cell workflow, the total RNA can be isolated and run with an Agilent TapeStation RNA Assay to evaluate the quality and to verify the total RNA is not degraded (RIN value ≥ 7 is recommended).
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