8. Detection of Expressed Barcodes or sgRNA with scRNA-Seq

CloneTracker XP™ Barcode Libraries and CRISPR sgRNA libraries cloned into Cellecta pScribe vectors (e.g. pRSGScribe1) express the barcode and/or the sgRNA guide on an RNA transcript (i.e., the selection marker gene) so it is readable in single cell RNA-Seq (scRNA-Seq) applications. As a result, the barcodes and/or guide sequences can be detected when running scRNA-Seq. It is only necessary to amplify the barcode and/or sgRNA sequence after making the cDNA.

The general approach to extract the barcode or guide RNA sequence is described below using the CloneTracker XP barcode as an example. The approach with the sgRNA libraries in Cellecta pScribe vectors is very similar. The specific protocol for both types of libraries can be found in the next Section: 10X Chromium 3’ Detection of Barcodes/sgRNA in pScribe Libraries.

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

For details on how to detect barcodes and/or guide sequence when using the 10X Chromium 3’-Protocol, see Section 10X Chromium 3’ Detection of Barcodes/sgRNA in pScribe Libraries.