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 3′ v2 chemistry, followed by supplementary information and supplementary protocols for other technologies or instruments.
- CITE-seq_and_Hashing_Protocol_180212 (protocol for performing CITE-seq and Cell Hashing in parallel)
- CITE-seq_Protocol_180212 (protocol for performing CITE-seq only)
- Cell_Hashing_Protocol_180212 (protocol for performing Cell Hashing only)
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′ v2 platform.
- Cell_Hashing_assay_scheme – Nucleotide-level illustration of Cell Hashing library generation on 10x Genomics single cell 3′ v2 platform.
- Antibody-oligo_Conjugation_Protocol_180212 In this protocol we summarize the steptavidin-biotin and iEDDA-based conjugation strategies.
- CITE-seq_on_ddSeq_Protocol Our supplementary protocol for performing CITE-seq on the Illumina / BioRad ddSeq system. This protocol is designed to be used in conjunction with our main protocol and the SureCell 3′ WTA library prep protocol from Illumina / Bio-Rad.