Cell Tracking by NGS Barcode Labeling

CloneTracker Lentiviral Barcode Libraries for Cell Labeling and Clonal Tracking

When investigating tumor progression, drug resistance, differentiation, development, hematopoiesis, and other related areas, there is considerable interest in understanding the heterogeneity of cell populations in both cultured and in vivo settings, by incorporating stable, heritable, and sequenceable barcodes in individual cells or cell populations.

• Assess how cell heterogeneity changes in response to drugs or other selections
• Analyze which traits allow cells to survive under different conditions
• Track how cell diversity changes with differentiation or disease progression

Label whole cell populations with stable, genomically integrated, single, identifiable, sequenceable barcodes for cell tracking and quantification of descendants in mixed downstream cultures or tissue. Library constructs integrate into the genomic DNA after transduction and can be detected by genomic DNA amplification and, with the CloneTracker XP libraries, in RNA sequencing analysis. Sub-libraries with defined barcodes are available to label several cell populations with distinct non-overlapping barcodes.

CloneTracker Pooled Lentiviral NGS Barcode Libraries

Cellecta's CloneTracker Libraries containing millions of barcodes encapsulated in VSV-G pseudotyped lentiviral particles efficiently transduce and integrate into the host cell genome of virtually any mammalian cells. When a cell population transduced at low number of viral particles to cells, individual cells pick up and insert a single barcode into their genomic DNA. As the host cells divide, the barcode sequences in the lentiviral vector will also replicate and will be passed onto daughter cells. Cells can be treated, grown for several passages, frozen and thawed, and the sequences within the lentiviral vector will remain in the host cell. After selection, treatment, or differentiation, just extract genomic DNA, amplify, and sequence to identify and quantify barcodes present in the selected cell population.

CloneTracker Pooled Lentiviral Expressed NGS Barcode Libraries

Cellecta’s CloneTracker XP Libraries have a configuration where the barcode is located at the 3′ end of the transcript so it is copied during cDNA synthesis, thus expressed and quantifiable using RNA-Seq. With this configuration, the clonal barcodes can be detected in single cell bead-based RNA expression profiling assays, such as 10X Genomics System, BD Rhapsody, or Dolomite Bio’s Nadia system, so that changes in gene activation in different clonal populations can be assayed over the course of an experiment to identify sub-populations of progeny with advantageous or harmful phenotypes relative to the specific conditions, such as increased drug resistance or sensitivity.

In addition to the CloneTracker XP Libraries containing a fluorescent protein marker like Venus, the CloneTracker XP-rLuc Library has a red-shifted luciferase (rLuc) derived from Photinus pyralis (Ppy RE9 mutant) as a reporter for bioluminescent imaging (BLI).

Applications

Single-cell RNA-Seq analysis is an important tool to analyze individual cell responses. However, important genes and pathways may not be active in all cells. Certain responses may only occur in a portion of cells, or different groups of cells can manifest divergent phenotypes. One way to address this challenge is to label individual cells with uniquely identifiable barcodes. Responses of each of the clonal progeny from this initial barcoded population can be assessed, and data correlating to how experimental conditions, differentiation, or developmental processes affect distinct groups of cells in a population can be extracted.

CloneTracker Barcode Cell Labeling Kits

Kits for labeling all cells in a particular population with a specific, single barcode are also provided. The barcode constructs developed by Cellecta are selectable by puromycin and -depending on which kit you choose- an optional red fluorescent protein (RFP) marker so cells containing barcodes will fluoresce red. Each barcode construct is designed so that the barcode can be differentiated by sequencing as well as by PCR and qPCR using barcode-specific primers. 
The progeny from each cell population can be sampled after differently labeled populations are co-cultured or otherwise grown together.

The CloneTracker Barcode Cell Labeling Kits are designed for labeling only a limited number of different cell populations. For studying clonal dynamics and growth heterogeneity in a complex cell population with mixed representation of different cell types (e.g. different by phenotype or genotype), we recommend Cellecta’s CloneTracker Libraries, see above.