We are continuously improving our protocols. Updates will be regularly posted here with detailed information on the improvements. Below are our main protocols for performing CITE-seq and Cell Hashing, specifically on Drop-seq or 10x Genomics single cell 3P chemistry (v2 and v3), followed by supplementary information and supplementary protocols for other technologies or instruments. These protocols can also be used on the 10x Genomics single cell 5P kit with the modifications described here. If you have specific questions, have a look at our FAQs.
- CITE-seq_and_Hashing_protocol_190213 (protocol for performing CITE-seq and Cell Hashing in parallel)
- CITE-seq_190213 (protocol for performing CITE-seq only)
- Cell_Hashing_protocol_190213 (protocol for performing Cell Hashing only)
02/2019: Minor edits on protocol compatibility to 10x version 3.
09/2018: We added a point-by-point covalent Antibody-Oligo conjugation protocol using iEDDA click chemistry. Minor edits and clarifications in the CITE-seq and Cell Hashing protocols: Note the reduced Tween concentration in staining buffer, reduced cDNA additive primer concentration, and optional addition of RNAse inhibitor to single cell RT buffer when super-loading. Added detailed description how to super-load and/or multiplex samples.
02/2018: We included a point-by-point Cell Hashing protocol, CITE-seq plus Cell Hashing protocol, and updated our antibody conjugation protocol.
10/2017: Addition of an antibody-oligo specific primer (cDNA additive primer) during cDNA amplification at low concentration significantly improves ADT library purity and yield in the subsequent ADT specific library PCR.
10/2017: We tested direct covalent antibody-oligo conjugation chemistries based on iEDDA click chemistry (including a commercially available kit) and observe comparable results with the indirect streptavidin-biotin coupling.
10/2017: We note that a cleavable linker adds no benefit in the streptavidin-biotin-linkage strategy and in case of using a direct conjugation chemistry, appears to be detrimental. When using streptavidin-biotin-linkage, we recommend now simply ordering biotinylated oligos.
- CITE-seq assay scheme – Nucleotide-level illustration of CITE-seq ADT library generation on 10x Genomics single cell 3′ platform.
- Cell_Hashing_assay_scheme – Nucleotide-level illustration of Cell Hashing library generation on 10x Genomics single cell 3′ platform.
- CITE-seq_Hyper_Conjugation_190321 This point-by-point protocol describes the covalent and irreversible conjugation of purified antibodies to oligonucleotides by iEDDA-click chemistry, essentially as previously described in Van Buggenum et al., 2016.
- Antibody-oligo_SAV_Conjugation_Protocol_180921 In this protocol we describe the steptavidin-biotin antibody-oligo conjugation strategy used in our proof-of-principle publication.