The BD™ AbSeq oligonucleotide-conjugated antibodies
The BD™ AbSeq oligonucleotide-conjugated antibodies, when incorporated into a single-cell mRNA-sequencing experiment, can yield combined transcript and protein expression data. Such data can provide vital insights into cellular processes and yield insightful information about gene expression. This article by Erickson, et.al provides a detailed experimental protocol for the combined analysis of 40 proteins and 400 genes on over 104 cells using the nano-well based BD Rhapsody™ platform. The protocol also includes a workflow for sample multiplexing for the unique identification of the source of the cell (Eg. tissue type or donor) in the downstream analysis following the upstream pooling.
Highlights of the protocol
The features of this unique protocol are:
- A step-by-step approach for obtaining both transcript and protein data in a single experiment.
- Considerations for data analysis.
- Steps for reduction of batch effects through sample multiplexing.
- Discussion of enrichment strategies of rare cell types.
The article describes these steps in detail:
- Defrosting preserved peripheral blood mononuclear cell (PBMC) samples.
- Preparing the BD Rhapsody™ cartridge.
- Performing sample multiplexing and BD™ AbSeq oligo staining.
- Loading of cells onto the BD Rhapsody™ cartridge and retrieval of cell captured beads containing mRNA and feature barcodes.
- Performing reverse transcription and Exo 1 treatment on the cell capture beads.
- Library preparation of multiplexed targeted mRNA, AbSeq, and sample tags.
- Analysing using a primary analysis pipeline.
The article also discusses the limitations of using this approach to assess protein expression, transcript expression, the number of cells that can be analysed, and enrichment. It also offers troubleshooting advice.
Read the article online here.