Single cell RNA sequencing (scRNA-seq) |
Manual or FACS isolation of single cells which are then lysed, undergo poly-A selection, cDNA preparation, and amplification and sequencing similar to bulk NGS (see above). Allows single cell resolution, but can be hampered by quality control limiting ability to compare cells. |
Stegle et al. (251) |
Imaging flow cytometry |
Combined antibody fluorescence with bright- and dark-field microscopy. This allows protein quantification and subcellular visualization. Limited number of channels can limit the ability to investigate large numbers of markers. |
McGrath et al. (181a) |
Mass cytometry (CyTOF)/HD flow cytometry |
High-dimensional flow cytometry techniques to assess large panels of antibodies. CyTOF utilizes metal isotope labels and mass spectroscopy, while HD cytometry uses photomultiplier tubes across multiple channels to allow a large number of fluorophores. |
Han et al. (113), Mair and Prlic (178) |
High-dimensional scRNA-seq |
Cell populations can be analyzed and sorted by CyTOF or HD cytometry. Isolated cells are then lysed and undergo single cell RNA-seq. Expense and similar issues associated with scRNA-seq can limit ability to apply in large cohorts. |
Stegle et al. (251) |
Assay of transposase-accessible chromatin sequencing (ATAC-seq) |
Tn5 transposase cleaves DNA fragments from accessible regions of the cell’s DNA. These sequences are then read and mapped to the chromosome. Areas with frequent reads show up as peaks, and these peaks are used to determine accessible regions of chromatin. |
Chang et al. (44) |
Chromatin immunoprecipitation sequencing (ChIP-seq) |
DNA binding proteins are crosslinked in vivo using formaldehyde. Chromatin is then digested and antibodies against the DNA binding proteins are used to precipitate out these proteins with their associated chromatin. The recovered chromatin is then sequenced and mapped. |
Davies et al. (57) |
Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) |
Antibodies with coupled oligonucleotide barcodes are able to bind to cell surface proteins. These cells then undergo scRNA-seq, and the unique barcode allows protein quantification in addition to RNA sequencing in the same readout. |
Stoeckius et al. (253) |
MethylC-seq |
Bisulfite treatment converts nonmethylated cytosine to uracil, providing a sequencing marker for methylation status of cytosine residues. This allows assessment of potential epigenetic changes in tissues and cells |
Urich et al. (266) |