Frequently Asked Questions

How many antibodies can you include in a single Phospho-seq experiment?

Because all of the antibodies are individually conjugated with complex 15 nt indices as part of the larger DNA-oligo, in theory you can use a near unlimited number of antibodies. I have used up to 100 antibodies in a single experiment with no problems in detection.

How do you choose which antibodies are compatible with Phospho-seq?

The primary criteria I use for selecting antibodies for use in Phospho-seq is whether they work in a) Intracellular Flow Cytometry (ICFC) or b) Immunocytochemistry (ICC). Both of these methods use fixed, permeabilized cells as in Phospho-seq, with ICFC being slighter more similar due to the necessary dissociation in both protocols. As with any other antibody based assay, Phospho-seq is affected by antibody sensitivity and/or background staining. Not all antibodies will work well with Phospho-seq, even those used in ICFC or ICC due to differences in fixation or permeabilization between individual protocols.

Is there a repository about which antibodies work well with Phospho-seq?

On this website, under the data tab, users can look at data from multiple experiments with different antibodies. I hope that these plots can indicate the utility of all antibodies I have used.

Are the conjugated antibodies compatible with other single-cell intracellular quantification assays?

Yes! Although I primarily use TSB tags (10X feature barcodes) for my antibodies due to potential RBP binding of TSA tags (Poly A), you can use the same conjugation reaction to add a TSA tag and then capture with any poly-A compatible technology like InCite-seq or NEAT-seq or by using Bridge Oligo A, you can capture with Phospho-seq as well.

Is Phospho-seq compatible with 10X Multiome (scATAC + scRNA) kits?

Yes it is, although when using TSB tags, the capture efficiency is lower because the ATAC modality is already being captured by a bridge oligo (the splint probe (10x Multiome Structure) ), so therefore you are using two bridges. If using TSA tags, you can directly capture like in NEAT-seq. It should be noted that running the multiome on fixed tissue results in significantly lower RNA data quality and we highly recommend using Bridge Integration (Hao et al., 2023) as an alternative if you have appropriate bridge and reference datasets available.

How long will the reagents included with the Phospho-seq kit last if stored properly?

In my experience the reagents are good for quite some time - I have tested conjugated antibodies stored at 4C and observed that they are still conjugated and functional after at least a year. The other reagents should be stable for at least that long. The one reagent that may be more limited in stability (closer to 6 months) is the TCO-labeled oligos. However, their instability manifests as the oligo losing its TCO-label very gradually. This means that where you may have added 15 pmol oligo per ug of antibody before, you may want to add 20 pmol if doing it 6 months later and 30 pmol if doing it a year later.