Technology

Our novel approach relies on Pre-templated Instant Partitions (PIPs) to simultaneously segregate complex cell mixtures into partitions with barcoded template particles that can be easily processed for single cell applications such as single cell RNA sequencing (scRNA-Seq). This approach (PIPseq™) eliminates the need for complex, expensive instrumentation and microfluidic consumables. 

Fluent BioSciences PIPseq workflow

1. Sample Preparation

  • Workflow beings with your prepared single cell suspensions which are mixed with our PIPs (templated core particles)
  • After the addition of a partitioning reagent, the cell and PIPs mixture is segregated into individual partitions through 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.