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

Highly Scalable Immune Cell Profiling in a Single Reaction

In this experiment, we processed ~200K frozen human PBMCs using the v4 chemistry T100 3’ Single Cell RNA Kit. The goals of this study were to showcase the unprecedented scale of the T100 kit and detail the ability to detect rare cell types in a single sample reaction. The sample was sequenced on an Illumina NovaSeq 6000 sequencer to a depth of ~2.6 billion reads (~18.5k reads per cell). Paired-end dual indexing was used with the following settings:

  • Read 1: 150 cycles
  • Read 2: 150 cycles
  • i5 index: 8 cycles
  • i7 index: 8 cycles

The sample was processed using PIPseeker v3.0.0 using –force-cells 140869. Cell type annotation was performed using the human-pbmc-v4-detailed annotation reference. A UMAP dimensionality reduction projection shows diverse cell populations at high resolution, showing the power of high cell input, single tube reactions. Importantly, the T100 kit resolved HSPCs, a very rare cell type in PBMCs that represent ~0.15% of the overall population.

Example RNA feature expression plots show clear localization for two HSPC markers, CD34 and SOX4.