Cell “hashing” with barcoded antibodies enables multiplexing and doublet detection for single cell genomics

Abstract

Despite rapid developments in single cell sequencing technology, sample-specific batch effects, detection of cell doublets, and the cost of generating massive datasets remain outstanding challenges. Here, we introduce cell "hashing", where oligo-tagged antibodies against ubiquitously expressed surface proteins are used to uniquely label cells from distinct samples, which can be subsequently pooled. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its sample of origin, and robustly identify doublets originating from multiple samples. We demonstrate our approach by pooling eight human PBMC samples on a single run of the 10x Chromium system, substantially reducing our per-cell costs for library generation. Cell "hashing" is inspired by, and complementary to, elegant multiplexing strategies based on genetic variation, which we also leverage to validate our results. We therefore envision that our approach will help to generalize the benefits of single cell multiplexing to diverse samples and experimental designs.

Normalizing and denoising protein expression data from droplet-based single cell profiling

“…We used CITE-seq Count 14 for HTO and ADT read mapping and Cellranger for UMI mapping. We demultiplexed cells as previously described 4,15,16 . UMAP 17 plots were generated with config parameter n.neighbors = 35, min.dist = 0.6.…”

Transcriptomics and single‐cell RNA‐sequencing

“…'Cell hashing' uses a different type of approach to label the exterior of a cell with a barcode so that individuals or samples with similar genetic backgrounds can be combined. 46 It is applicable to genetically identical experimental mouse strains as well as samples taken from related individuals. These multiplexing techniques form the basis of future population-scale single-cell genomics studies, and additional methods based on CRISPR-Cas9 gene editing are likely to follow.…”

Expanding the CITE-seq tool-kit: Detection of proteins, transcriptomes, clonotypes and CRISPR perturbations with multiplexing, in a single assay

“…To enable profiling of protein markers together with V(D)J regions and transcriptomes, we modified our previously described CITE-seq method 11 . Oligos partially complementary to the gel bead-associated template switch oligos in the 10x Genomics 5P / V(D)J kit were covalently conjugated to antibodies as described 13 and used to label cells. Annealing and extension during the reverse transcription (RT) reaction appends the cell barcode and unique molecular identifier (UMI) to each oligo in parallel with the addition of these sequences to the first strand cDNA copies of cellular mRNAs in the same droplet ( Figure 1a) (see methods).…”

“…Antibodies used for CITE-seq and Cell Hashing were obtained as purified, unconjugated reagents from BioLegend (see Supplementary Tables 1, 3 & 4) and were covalently and irreversibly conjugated to barcode oligos by iEDDA-click chemistry as previously described 13 .…”

“…After final elution, separate PCR reactions were set up to generate the ADT library (SI-PCR and RPIx primers), the hashtag library (SI-PCR and D7xx_s) and the GDO library (SI-PCR and Next_nst_x). The ADT and HTO libraries were prepared as previously described 13…”