6. Detection of Expressed Barcodes Using Single-Cell RNA-Seq

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 in the Barcode-3’ Libraries is located in the 3’-dLTR (3’-UTR) region of the reporter/drug resistance marker transcript. In the Barcode-5’ Libraries, the cassette is located immediately downstream of the promoter driving the reporter and marker expression.

The barcode has a composite structure built by positioning of multiple 14-18-nt sequences with multiple 30-nt sequences separated by a short spacer (4nt spacer in the 1M/10M libraries and 21nt spacer in the 5M/50M libraries). With the Barcode-3’ libraries, the barcode cassette is positioned within approximately 90 nt of the polyA start of the transcript while the barcode is approximately 150-200 nt from the polyA start (depending on the particular vector). 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 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.

Linear map of pScribe-EFS-TagRFP-2A-Puro-BC14-BC30-3LTR

Using the CloneTracker XP Barcode-3’ Library with the 10x Genomics Chromium Kits

The 10x Chromium 3’ Reagent Kit was developed for amplification of 3’-ends of polyA+ RNAs and based on ligation of 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 100 bases or so. The CloneTracker XP BC14-spacer-BC30 barcode is located at a fixed ~150-200nt distance upstream from the poly-A+ site, depending on the particular vector. 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 barcode sequence in the CloneTracker XP Barcode-3’ Library when using the 10x Genomics Chromium 3’ Kit, you need to include an additional PCR amplification step with a barcode-specific primer that is located just upstream of the CloneTracker XP barcode. This will ensure amplification of the segment of cDNA that contains both the CloneTracker XP barcode and the cell barcode associated with the poly-A+ sequence in the scRNA-Seq protocol (e.g., the “bead-barcode” with bead-based scRNA systems). The general procedure for how to integrate this additional PCR with the 10x Chromium 3’ Kit procedure is shown below.

Please note that we can only provide general guidelines. Depending on the specific scRNA-Seq protocol you are using, some modifications and optimization will be required.

Set up a separate PCR reaction with a portion of cDNA just after the cDNA amplification/cleaning/quantitation step (Step 2.4, just before the cDNA fragmentation step), since the 10x protocol only needs ~25% of cDNA for profiling.

1. Take another 25% (10 ul) of amplified cDNA, and specifically amplify barcode using a combination of Partial Read 1 primer (5’-CTACACGACGCTCTTCCGATCT-3’ — see 10x Chromium 3’ Kit User Guide) and Adaptor-FBP1:

                                  Adaptor (Read 2)                                             FBP1 primer
5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCGACCACCGAACGCAACGCACGCA-3’

• The standard Illumina adaptor sequence is located in the first 34 nucleotides of the 5’ end (underlined).

* Note: Other scRNA-Seq protocols may use a different adaptor, in which case you should modify the primer accordingly.

2. Amplify both barcode and adaptor-ligated cDNA (generated at Step 3.5 in 10x Chromium 3’ protocol) samples using reagents from kit and the same program, but add an additional 6 cycles for barcode sample. The 6 additional PCR cycles will increase the yield of the CloneTracker XP barcoded cDNA sequences to approximately 64-fold higher levels than other transcripts in the cDNA reaction. This helps ensure you recover the majority of the CloneTracker XP barcodes.

3. Run a Bioanalyzer analysis after amplification. You should be able to see a band of ~350-375nt (depending on the particular vector), which corresponds to the barcode product amplified from lentiviral barcoded transcript. The amplified product will have the following structure—Read2(Adaptor)-FBP1-Barcode-UMI-10xBarcode-Read1—which is exactly the same as PCR products generated from amplified adaptor-ligated cDNA—Read2(Adaptor)-cDNA-UMI-10xBarcode-Read1. If you can’t see the barcode-specific product, add an additional 2-4 extra cycles and repeat Bioanalyzer analysis.

4. After this step, then amplify the adaptor-ligated cDNA and barcodes using different i7 and i5 index primers, quantitate them, purify using SPRI beads, and mix at the desired ratio (e.g. 9:1) for the follow-up NGS step.