Fluent BioSciences PIPseq workflow
1. Sample Preparation
- Workflow beings with your prepared single cell suspensions which are mixed with our PIPs (templated core particles that have unique barcodes to capture cell and transcript information)
- After the addition of a partitioning reagent, the cell and PIPs mixture is segregated into individual emulsions by vortexing
- Cells are then lysed on a thermal device and their mRNA captured onto the PIPs
- cDNA is then generated via reverse transcription (RT) and amplified via standard PCR
2. Library Preparation
- cDNA libraries are generated via fragmentation, end repair and A-tailing
- This is then followed by adapter ligation and cleanup
- Sample indices (compatible with Illumina sequencing) are then added, followed by a final cleanup
3. Next generation sequencing (NGS)
- Libraries can then be processed on the appropriate Illumina NGS instrument.
- The sequencing reagent kit (and chip) is chosen based on the number of samples multiplexed, cell count and desired read depth
4. Data Analysis
- The Fluent Cloud Platform enables primary analysis of the PIPseq sequencing libraries.
- Upload your FASTQ files into the portal which are then processed into standard feature-barcode count matrices, along with summary metrics (such as genes and UMIs per cell) and diagnostic plots.
- Note: Count matrices are provided in a standard format compatible with widely-used open-source secondary analysis software packages.
Select from the below resources to learn more about our protocols (user guides) or customer applications (posters).