Setting Up for Experimental Success: Exploring the Importance of Pilot Studies


Our novel approach relies on Particle-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

  • During sample preparation, cell suspension of interest is mixed with our core template particles and segregated into Particle-Templated Instant Partitions (PIPs) by simple vortexing
  • The cells in PIPs are then lysed on a thermal device and the mRNA is captured by barcoded oligonucleotides incorporated with the template particles

2. Library Preparation

  • The captured mRNA undergoes reverse transcription and cDNA libraries are then 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 PIPseekerTM Software enables primary analysis of the PIPseq sequencing libraries 
  • Upload your FASTQ files into the software and obtain summary metrics, diagnostic plots, clustering and differential gene expression tables
  • Also generate standard feature-barcode count matrices, which are compatible with widely-used open-source tertiary analysis packages