AbSeq Protocol Using the Nano-Well Cartridge-Based Rhapsody Platform to Generate Protein and Transcript Expression Data on the Single-Cell Level

Abstract

By including oligonucleotide-labeled antibodies into high-throughput single-cell RNA-sequencing protocols, combined transcript and protein expression data can be acquired on the single-cell level. Here, we describe a protocol for the combined analysis of over 40 proteins and 400 genes on over 10⁴ cells using the nano-well based Rhapsody platform. We also include a workflow for sample multiplexing, which uniquely identifies the initial source of cells (such as tissue type or donor) in the downstream analysis after upstream pooling. For complete information on the use and execution of this protocol, please refer to Mair et al. (2020).

Graphical abstract

Figure 1

Setting Up the BD Rhapsody Express BD Rhapsody Express showing proper cartridge position with 5 mL LoBind Tube for collection of Cell Capture Beads and waste container installed. During the workflow, the slider needs to be moved either to “Waste” or to “Beads” as indicated in the protocol.

Figure 2

Loading the Cartridge Insert the pipette perpendicular to the cartridge with enough pressure to properly seal the orange gasket with the pipette tip, then dispense the pipette contents. The pipette needs to be set to “Prime/Treat”.

Figure 4

BD Rhapsody Set to Retrieval When the left slider is set to RETRIEVAL, the top magnet will sit atop the cartridge.

Figure 5

Bead Collection After cell lysis, collect the mRNA and oligo-bound beads using the P5000M pipette. Make sure that the magnet is in the correct position and the tube slider is set to “Beads”.

Figure 6

Washing Cell Capture Beads Allow beads to settle next to the magnet, and remove lysis buffer without disturbing the beads.

Figure 3

Loading the Cell Capture Beads Hold pipette vertically and with enough pressure to seal the gasket while quickly dispensing beads to avoid settling. The pipette needs to be set to “Bead Load”.

Figure 7

Library Preparation Workflow Overview of each PCR required for library preparation. After PCR1, DNA double-sided size selection will split the transcripts based on amplicon size into mRNA or Sample Tag/AbSeq libraries. PCR2 is then preformed to further enrich both mRNA and Sample Tag libraries. Finally, each library is indexed for sequencing.

Figure 8

Example of AbSeq/Sample Tag PCR1 Products The largest peak should be approximately 170 bp. Other base pair lengths present are indicative of incomplete double-sided selection.

Figure 9

Example of Final mRNA Library TapeStation Trace The major peak length will depend on the PCR panels used. If you detect some transcripts with longer base pair lengths, this is typically not an issue as shorter transcripts amplify more efficiently than longer products.

Figure 10

Example of Final Sample Tag Library TapeStation Trace The expected size of the final Sample Tag library is approximately 290 bp. If you observe a smaller peak (around 270 bp), it is likely those transcripts are AbSeq products.

Figure 11

Example of Final AbSeq Library TapeStation Trace The expected size of the AbSeq final library is around 270bp.

Figure 12

Example of a Poor-Quality Trace This is an example of a library that had an incomplete double-sided selection. The library should undergo another round of cleanup.