Frequently Asked Questions

If you have questions, comments or suggestions that are not addressed, here please contact us.

CITE-seq count FAQ. Can be found in the GitHub wiki.

Should I use Streptavidin-Biotin conjugation or direct covalent conjugation of antibodies to oligonucleotides? Antibody-oligo conjugates can be prepared by various methods which form covalent links between antibodies and oligos (e.g. amine-reactive NHS or iEDDA-, Maleimide- click chemistries) or by indirect streptavidin-biotin-linkage. For our proof- of-principle experiments we used streptavidin-biotin-linkage to couple oligos to antibodies, and include a cleavable linker in the oligos. We are now using direct antibody-oligo conjugation chemistry (iEDDA, essentially as described in Van Buggenum et al., Scientific Reports, 2016; without a cleavable linker), and antibody-oligo conjugates from BioLegend. We have compared direct and indirect conjugation methods and had very comparable results. Direct conjugation has the added benefit that larger panels can be pooled and stored for prolonged periods of time.

How many antibodies can be multiplexed in one CITE-seq experiment? The DNA-barcode on the antibody-oligos allows a virtually ‘limitless’ number of barcodes, far exceeding the numbers of fluorophores for flow cytometry or isotopes for mass cytometry. We have successfully used up to ~120 antibodies and do not foresee any reason why this number can’t be further increased. We have also not observed any competition between mRNAs and Antibody-oligos for polyT primers, larger panels and high concentrations of Cell Hashing antibodies did not result in decreased nUMI or nGene counts.

Why did you chose a small RNA read 2 PCR handle on the CITE-seq antibody-oligos?  We wanted to keep the oligos on the antibodies as short as possible to avoid potential adverse effects on antibody specificity or accessibility. Small RNA read 2 handle is currently the shortest sequencing library handle from Illumina without partial reverse complementarity to read 1, as for Truseq DNA or Nextera primers. Illumina sequencers run with a pool of all possible illumina sequencing primers so the machines are agnostic to the primers used in a particular library pool. Therefore ADT/HTO and cDNA libraries can be pooled without any alterations to the standard sequencing protocol.

What do the “xxxx” mean in the RPI-x and TruSeq D7xx_s primers?  The “x” nucleotides are meant to indicate that you can use whatever sequence you want in these positions.  Standard index sequences from Illumina work, as do custom sequences.  The 10x i7 indexes used for indexing your 3′ tag cDNA libraries are a pool of 4 separate indexes per sample, so be sure to check that the indexes you use for your ADT / HTO libraries have sufficient edit distance from these indexes and from each other.  Note that the original Illumina RPI-x primers have 6 nt indexes.  If you are using these together with 8 nt indexes, the sequence of your 6 nt index, as read by the sequencer is “xxxxxxAT”.

What is with the “_s” suffix on the TruSeq D7xx_s primers?  The “_s” suffix indicates that these primers are “short”.  Their 3 end is right at the start of the 12 nt stretch that is common to TruSeq read 1 and TruSeq read 2 primers.  The “_s” primers were designed this way to prevent mis-priming on the read 1 end of the HTO library. Libraries made with these _s primers are not truncated in any way, they will still sequence normally on all machines.  Refer to the Cell_Hashing_assay_scheme for more information.  We have seen better performance with these D70X_s vs. D701 primers but also never went back to really quantify it.

Where should I order the primers described in the protocol and what should they be diluted in? We order our oligonucleotides at any common oligonucleotide synthesis company. We typically dilute our oligo stocks at 100 micromolar in water or TE, and have our working concentrations as described in the protocols.

Do any of the oligos for the protocol have to be ordered PAGE or HPLC purified?  This entirely depends on the lab budget. We typically do not order our oligos HPLC or PAGE purified, simply desalted oligos work well.

Why are you adding ADT and/or HTO additive primers to the cDNA amplification?  Addition of an antibody-oligo specific primer (cDNA additive primer) during cDNA amplification at low concentration significantly improves ADT and/or HTO library purity and yield in the subsequent ADT specific library PCR. We add these primers in very low concentration not to interfere with the amplification of the full length mRNA cDNAs.

CD45 vs. CD298 & B2M Hashing Antibodies. For our proof of principle Cell Hashing experiments using PBMCs we have used multiple ‘ubiquitous’ immune markers including CD45. We have now entirely switched to using a mix of CD298 and B2M, which are expressed on a large variety of tissues and cell types, including immune cells. This allows Cell Hashing of virtually any sample, which might contain mixtures different celltypes.

My ADT (or HTO) library contains a large fraction of RT-TSO dimer (~150nt). The RT-TSO dimer is carried over from the cDNA amplification and is typically dwarfed simply by doing 1 or 2 additional cycles of ADT PCR on the purified ADT library. Carryover of cDNA amplification primers will re-amplify the RT-TSO dimer and mRNA-cDNA fragments in the ADT library, if this is a recurrent problem try purifying your ADT library with two rounds of 1.8X SPRI (instead of 2X SPRI). Also, this is typically a bigger problem with very small antibody panels (less than ten), the larger your antibody panel gets the less prominent this dimer will be.

My ADT (or HTO) library contains a large broad peak at around 400 bp. Excessive library amplification leads to exhaustion of primers (and/or dNTPs) which results in “daisy-chains” or “bubble products”, heterogenous library products that anneal by their P5 and/or P7 adapter sequences. These appear as very characteristic high molecular weight smooth DNA peaks typically around double the size of the correct product and can be more or less prominent (panel A,B below). This larger peak will cluster and sequence perfectly and it will be also a good quality library. But it is very hard to accurately quantify these libraries. If you encounter a bubble product in your library you can get rid of it by simply performing another PCR reaction (using same primersets as first PCR or P5/P7 generic primers) for one or two cycles on the 1.6X SPRI cleaned up product. Depending on the amount of ‘daisy-chained’ product, these additional PCR cycles will convert the library partially (panel C) or entirely (panel D) to double stranded DNA, collapsing the product size to the correct size range.


I do not observe a ADT (or HTO) product on the Bioanalyzer in the supernatant fraction after cDNA amplification. We typically never run the ADT/HTO-supernatant fraction on the BA, there is usually nothing visible from the ADT (or HTOs)! This fraction typically only contains a very prominent ~100 band from RT-TSO hybrid (see assay our assay scheme). After the ADT/HTO PCR there should be a clear and sharp product at ~180 bp.

What are P5 and P7 generic primers? These primers anneal to the P5 and P7 regions of Illumina libaries and can be used to re-amplify any Illumina sequencing library.  P5_generic:  5’AATGATACGGCGACCACCGAGATCTACAC, P7_generic: 5’CAAGCAGAAGACGGCATACGAGAT

Are 5P CITE-seq and 5P Hashing antibodies commercially available? As far as we know, 5P compatible antibodies for CITE-seq and Hashing will be commercially available soon, although we don’t know the exact timing. We’d suggest reaching out to BioLegend, who may be able to provide a more solid timeline.

I want to run FACS prior to CITE-seq. Can I stain samples with Cell Hashing and/or CITE-seq and our fluorescent panel at the same time? Yes. We often first enrich cell populations by FACS and then run CITE-seq or multiplex different sorted samples by Cell Hashing. To maximize viability and eliminate multiple staining/incubation steps we typically stain the cells with CITE-seq and/or Cell Hashing and flow cytometry antibodies at once before the sort and then go directly into CITE-seq after the sort. Although the sort also acts like a pretty good wash we would still wash the cells twice before going into the scRNA-seq run.