CloneTracker XP™ Barcode Libraries express a barcode (i.e., a unique molecular identifier or UMI) as an RNA transcript readable in single cell RNA-Seq (scRNA-Seq) applications. As a result, scRNA-Seq in combination with RNA expressed barcodes allows you to both identify and phenotypically characterize the different barcoded cell lineages across highly diverse cell populations.
The lentiviral-based CloneTracker XP Barcode Libraries have two main functional elements—the reporter (RFP, Venus, or rLuc) and drug resistance marker (PuroR)—both expressed from one promoter on a single transcript. The barcode cassette is located in the 3’-dLTR (3’-UTR) region of the reporter/drug resistance marker transcript. The 48-nucleotide barcode has a composite structure built by juxtapositioning 100 different 14-nt sequences and 100,000 30-nt sequences separated by a 4-nt spacer (TGGT). It is positioned within approximately 70 nt of the polyA start of the transcript. As a result, the transcribed barcodes are captured by cDNA first strand synthesis using barcoded oligo-dT primers, which is the typical first step of most scRNA-Seq procedures (e.g. Drop-Seq, 10x Genomics, BD Rhapsody, Bio-Rad ddSeq, Takara SMARTer ICELL8 system, etc).
Each composite barcode (14-nt + TGGT + 30-nt sequence) is a unique and highly diverse sequence that tolerates up to 5 mutations that help ensure accurate identification of barcodes even in the most demanding applications (e.g., cell lineage tracing in vivo). A link to the list of barcode sequences for your library is provided with the Product Information emailed to you upon shipment of the library. Please contact Cellecta if you did not receive this email.
Amplifying the CloneTracker Barcode in scRNA cDNA
Most scRNA-seq protocols add an adaptor upstream of cDNA at a variable distance from the poly-A+ priming site. The ligated adaptor is used as a binding site for a primer to read into the mRNA coding sequence. The sequence read from this upstream adaptor primer is typically only just a 100 bases or so. The CloneTracker XP BC14-TGGT-BC30 barcode is located at fixed 70-nt distance upstream from poly-A+ site. Depending on the distance of the adaptor from the poly-A+ primed site, the cDNA, the CloneTracker XP barcode will often be out of reach of the adaptor sequencing run.
To effectively read the CloneTracker XP barcode sequence with most single cell protocols, you need to include a PCR with a barcode-specific primer that is just upstream of the CloneTracker barcode. This additional primer will ensure amplification of the segment of cDNA that contains both the CloneTracker barcode and the cell barcode associated with the poly-A+ sequence in the scRNA protocol (e.g., the “bead-barcode” with bead-based scRNA systems). Without this modification, CloneTracker barcodes with adaptor primers located too far upstream will not be captured and, therefore, be poorly represented.
The additional PCR to amplify the segment of the cDNA with the CloneTracker barcode and the cell barcode (on the poly-A+ end), can run in either of two ways:
Option 1: Include CloneTracker Barcode PCR with Single-Cell cDNA Preparation: The CloneTracker Barcode PCR can be incorporated into the single-cell protocol by adding the CloneTracker barcode PCR primer, with the scRNA upstream adaptor primer, into the adaptor primer PCR. This is a convenient approach but if the scRNA procedure produces a low number of reads per cell, the yield of the CloneTracker barcode sequence may be too low to reliably detect for all cells.
Option 2: Separately Amplify CloneTracker Barcodes: The CloneTracker Barcode PCR can be run as a separate PCR on the adaptor-ligated cDNA products from the scRNA protocol. This approach does not require any changes to the scRNA protocol other than using some of the cDNA preparation for the CloneTracker PCR. Since it is a separate PCR reaction, the yield for the CloneTracker transcript relative to the rest of the cDNA can be increased by increasing the number of PCR cycles in this reaction.
The primers and guidelines for both approaches are described in the follow section. Please note that we can only provide general guidelines in more detail in the next section. Depending on the specific scRNA protocol you are using, some modifications and optimization will be required.
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