There are only four reverse primers (four indexes) provided in the kit. Can I use more than four reverse index primers?

Yes, more than four indexes can be used at a time. The additional primers can be ordered through the IDT TruGrade service and are HPLC purified and resuspended in IDTE pH 8.0 at a 10 μM concentration. See technical bulletin Ordering Additional Indexes for the BD Rhapsody™ Library Reagent Kits  for more information.

Can the BD Pharmingen™ Stain Buffer be substituted?

The BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) is the only recommended stain buffer. All other buffers have not been tested.

Can cells be loaded with media instead of sample buffer?

Cells can be loaded in PBS plus up to 1% BSA, but a large volume of old cell media can have a negative impact on bead-loading efficiency.

Can fixed cells be used?

We do not support a protocol for fixed cells at the moment. However, there have been publications on single-cell mRNA applications with methanol fixed cells (https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-017-0383-5#Sec2). Another fixative that preserves mRNA and has been shown to work with other single-cell platforms is CellCover. These methods may work with the BD Rhapsody™ System. Note that calcein only stains live cells. For visualization of fixed cells on the BD Rhapsody™ Scanner, you can try green nuclei stain or strong green stain for dead or fixed cells (https://www.thermofisher.com/order/catalog/product/L23101). We do not anticipate FFPE and formaldehyde fixed cells to work with the BD Rhapsody™ System as mRNA are heavily fragmented by such fixation methods.

How stable is the BD Rhapsody™ Cartridge after it has been removed from the foil package (before priming step)?

The BD Rhapsody™ Cartridge is stable for 24 hours after removal from the foil package.

How critical is it to keep lysis time to 2 minutes?

It is very critical since longer lysis times can lead to mRNA cross-hybridization between cells (indicated by low % reads to putative cells sequencing metric).

How do I calibrate automated pipettes?

Pipettes can be sent to Gilson directly for calibration. Additional information: Lead time is 3–5 days depending on the facility. Yearly service and calibration are recommended.

How can I run multiple cartridges in parallel?

The BD Rhapsody™ Single-Cell Analysis System Instrument User Guide provides a recommended protocol for running two cartridges side by side with one sample loading station.

How are doublets identified? 

Cells are stained with Calcein AM. Individual cells are identified based on fluorescent imaging, and doublets or multiplets that are clustered together are detected based on the shape or eccentricity and variation in fluorescence intensity within the cluster. Doublets in wells are determined by overlaying fluorescent imaging of live cells and brightfield imaging of the wells in the cartridge.

How does bead subsampling work?

Beads with synthesized cDNA can be aliquoted as needed and amplified to create a sequencing library. For instance, if the cartridge captures 10,000 cells, proceeds to cDNA synthesis and stores the beads, you can take 10% of the stored beads to make a sequencing library representing ~1,000 cells.

If the diversity of UMI is only ~65,000, does it mean you can only capture a total of ~65,000 molecules?

No, the diversity of UMI applies to a gene of a cell not for all genes across a cell and not for all genes across all cells. The definition of a molecule is a unique combination of a UMI sequence, a gene and a cell label. In a typical cell, for a gene, we see a copy number of zero to at most a few thousand. Thus, an UMI diversity of ~65,000 is more than sufficient to ensure each molecule is tagged uniquely.