Frequently Asked Questions
If you have questions, comments or suggestions, please contact us.
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 90 antibodies and do not foresee any reason why this number can’t be further increased.
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. Refer to the Cell Hashing assay scheme for more information.
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.