By transducing the CloneTracker or CloneTracker XP Barcode Library into a large pooled cell population, you can create a founder population in which each cell contains a unique integrated barcode. During transduction, the library of lentiviral constructs carrying each barcode enter the cells and stably integrate into the genomic DNA. Each lentiviral construct also has an RFP marker and puromycin selection to help maintain the barcode cassette.
Notes on Transducing Founder Cells
- Cell transduction is a random process following a statistical distribution. Therefore, if too high an MOI is used, many cells will take up more than one barcode. Cells with more than one barcode show up as more than one population in the final analysis. For example, if two barcodes integrate into one founder cell, and the cell produces 50 progeny, the data (after harvesting the cells and Next-Gen Sequencing of the genomic DNA) will show two clonal populations with two different barcodes, each having the same number of cells. It will not be obvious that these two populations are from the same founder cell. For this reason, we typically recommend using low MOIs of <0.2 so that >90% of the transduced cells only contain one barcode.
- Transducing larger populations of cells increases the frequency of having more than one founder cell with the same barcode. Since the CloneTracker Barcode Library has several million unique barcodes, a majority of cells will contain unique barcodes, even with library transductions of a million or more. However, with larger transductions, two or more founder cells can receive the same barcode. To minimize this, we recommend starting experiments with fewer than a million cells for libraries with 10M or more barcodes, and fewer than 300,000 cells for libraries with 1-5M barcodes, if possible. For more details on the complexity and representation of barcodes in the library, and estimates of the number of barcodes repeats you should expect with transductions of different size founder cell populations, please refer to the QC information available on the PAC or described in the Quality Control section.
- Since the purpose of using the CloneTracker Barcode Library is to track the fate of cells from a founder population, we do not recommend splitting and discarding any cells during the course of an experiment:
- Discarding any portion of cells will eliminate some of the barcodes unless the cell population has expanded to several orders of magnitude over the founder population size.
- Even if the population has expanded many fold over, splitting or otherwise discarding cells may skew the clonal population sizes for different barcodes (i.e., clones that grow slowly versus cells that grow quickly may be differentially affected).
- If you must discard cells, it is crucial to design the experiment to minimize the impact on the barcode representation in your samples.
- Following transduction, you may want to select cells with barcodes using puromycin. To empirically determine the concentration of antibiotic that kills untransduced cells within a given amount of time, you should calculate a Puromycin Kill Curve as described in the Recommended Pilot Experiments section.
In this section of the manual, you will find step-by-step protocols for general packaging, transducing, and titering lentiviral plasmid libraries and constructs.
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