AbSeq Protocol Using the Nano-Well Cartridge-Based Rhapsody Platform to Generate Protein and Transcript Expression Data on the Single-Cell Level
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(TM) Single-Cell Analysis System. 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(TM) AbSeq Ab Oligos 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.
Read the paper “AbSeq Protocol Using the Nano-Well Cartridge-Based Rhapsody Platform to Generate Protein and Transcript Expression Data on the Single-Cell Level”
