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

Surface Protein Epitope Labeling in PBMCs

In this experiment, 40k PBMCs from a healthy human donor were labeled using BioLegend TotalSeq-A Universal antibody panel and processed using the PIPseq v4 chemistry T20 3’ Single Cell RNA Kit. The add-on Epitope Sequencing Kit was used to process the ADT library. The goal of this experiment was to demonstrate compatibility with CITE-seq applications and quantify the power of these protein-derived tags to support identified cell types. RNA samples were sequenced on an Illumina NextSeq 2000 sequencer to a depth of ~367 million reads (~14k reads per cell). ADT samples were also sequenced on an Illumina NextSeq 2000 to a depth of ~257 million reads (~9.8k reads per cell). Paired-end dual indexing was used for both libraries with the following settings:

  • Read 1: 54 cycles
  • Read 2: 67 cycles
  • i5 index: 8 cycles
  • i7 index: 8 cycles

Samples were processed using PIPseeker v3 using –force-cells 26219, including designation of ADT-specific parameters. Cell type annotation was performed using the human-pbmc-v4-detailed annotation reference. Standard PBMC cell types were identified, plus Naive CD8 T and Plasmacytoid Dendritic Cells (pDCs). ADT library quality metrics and individual ADT feature expression plots were output by default in PIPseeker. 

Example ADT feature expression plots exhibit clear localization for T cells (CD3D), CD14 Monocytes (CD14), and B cells (CD19)