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.

Major updates:

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.

Supplementary information:

  • 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.

Supplementary protocols: