Cell hashing is a technique used in single-cell experiments to multiplex samples prior to encapsulation and/or lysis steps, which can help reduce overall reagent usage and sequencing costs. We offer TotalSeq™-A, -B, -C, and -D hashtag formats that contain 1:1 mixtures of two abundant surface markers for this purpose, CD45 and MHC I for mice, and CD298 and β2 microglobulin for human samples. In addition to single-cell analysis using intact cells, single-nucleus analysis makes it possible to characterize cellular states and physiology in tissues that are challenging to dissociate. This includes tissues that are rich in certain cell types like neurons, adipocytes, and muscle cells. Single-nucleus analysis can also help when tissue storage is required, as it is difficult to recover and obtain single-cell suspensions from archived frozen material. For experiments where nuclei are the input sample type, we offer TotalSeq-A, -B, and -C Anti-Nuclear Pore Complex Proteins Hashtags, which have been successfully used to multiplex samples in single nucleus RNA sequencing (snRNA-Seq) and intranuclear cellular indexing of transcriptomes and epitopes sequencing (inCITE-Seq) experiments. These multimodal assays allow for simultaneous profiling of transcriptomics, proteomics, and chromatin accessibility, helping to resolve the clustering of rare cell types due to the improved resolution of cell states.

 

These nuclear hashtags consist of a barcode oligo conjugated to clone MAb414, which is specific for p62, a serine/threonine-rich protein of ~520 amino acids, with tetrapeptide repeats on the amino terminus and a series of alpha-helical regions with hydrophobic heptad repeats. p62 assembles into a ~235 kDa complex containing three additional proteins, p60, p54, and p45. Glycosylation appears to be involved in the assembly and disassembly of p62 into higher-order complexes, and a serine/threonine-rich linker region between Ser270 to Thr294 appears to be regulatory. The p62 complex is localized to both the nucleoplasmic and cytoplasmic sides of the pore complex, and the relative diameter of p62 complex relative to the nuclear pore complex suggests it interacts in pore gating.

 

Just as viability is crucial for optimal protein staining in CITE-Seq, the quality of nuclei after isolation is critically important for nuclei-based assays. However, nuclei isolation can require a significant amount of optimization depending on the starting tissue and intended downstream analyses. Some general guidelines for handling nuclei and successful staining are outlined below:

 

• Lysis times should be optimized for each sample type. Typically, primary patient samples will require shorter lysis times than cell lines. Over-lysing samples will result in poor nuclei quality or, in the worst-case scenario, destroy the nuclei. Under-lysing cells will inhibit the transposition reaction.
• Nuclei are fragile. Nuclei tend to be sticky once isolated, so care should be taken especially when resuspending pellets to avoid shearing. Nuclei should be kept on ice at all times. Additionally, it is important to handle the nuclei carefully and optimize methods like sorting since extensive and harsh handling can potentially damage the nuclear membrane, leading to leakage of nuclear content.
• Assess nuclei quality visually. Many nuclei isolation protocols recommend assessing nuclei quality visually, using either a cell counter like a Countess or a microscope. Nuclei should appear round and intact without signs of blebbing and will stain as dead with a viability dye. Nuclei can also be sorted using DAPI, but quality should still be assessed visually before proceeding with any workflow.
• If the goal is to generate scATAC data, nuclei isolation optimization can be assessed using bulk ATAC-seq. Shallow sequencing of bulk ATAC-seq libraries is generally sufficient for determining the success of the optimization protocol and has the benefit of providing pilot data. A simple but robust ATAC-seq protocol can be found here.
• Nuclear hashtags must be titrated. As with any antibody, it is important to determine successful binding and optimal staining concentration prior to use with precious samples. Nuclear hashtags can be titrated via flow using fluor-conjugated versions of MAb414 on optimally-isolated nuclei, and additionally observed with a fluorescent microscope.

 

FAQs

What are nuclear hashtags?

Nuclear hashtags are used for single-nucleus RNA-seq (snRNA-seq) samples. snRNA-seq captures RNAs that are isolated with the nucleus. This is done because whole, intact cells may be difficult to isolate due to specific experimental or sample conditions, or to answer a specific scientific question. See an example of nuclear hashtag application in this paper.

 

Can I use nuclear hashtags for single-cell ATAC-seq?

Unfortunately, our TotalSeq™ reagents are not directly compatible with single ATAC-seq kits at this time. However, the NYGC has pioneered a bridging method ATAC with Select Antigen Profiling by sequencing (ASAP-seq) which is able to bridge TotalSeq™ antibodies with other capture platforms such as scATAC-seq. Our nuclear hashtag products are available off-the-shelf, but we do not currently have a recommended protocol for this application at this time. The only recommendations we can provide are literature references such as this one.

 

For additional guidance and information, please see the following resources:

 

10x Genomics Nuclei Isolation from Cell Suspensions and Tissues for Single Cell RNA Sequencing

Nuclei isolation for inCITE-seq

TotalSeq™-B or -C with 10x Feature Barcoding Technology

 

Nuclei isolation for snRNA-seq:

 

Protocol for Flow Cytometry-Assisted Single-Nucleus RNA Sequencing of Human and Mouse Adipose Tissue with Sample Multiplexing

Molecular Connectomics Reveals a Glucagon-Like Peptide 1 Sensitive Neural Circuit for Satiety

Nuclei Multiplexing with Barcoded Antibodies for Single-Nucleus Genomics

A Single-Cell and Single-Nucleus RNA-Seq Toolbox for Fresh and Frozen Human Tumors