Differential expression (DE) analysis focuses on comparing the relative expression levels of different transcripts in the cell and is one of the primary analysis tools used to explore transcriptome variability. End-capture methods are appealing for DE analysis as they can decrease the number of reads necessary to determine differential expression between cells. By focusing the sequencing data on a portion of each transcript (in this case, the 3' end of each transcript), we can reduce the number of reads and hence the overall cost required to identify expressed genes. Additionally, samples can be pooled prior to sequencing, decreasing the work and resources required and increasing the multiplexing capabilities of each sequencing lane.
In this tech note, we demonstrate the use of SMART-Seq v4 chemistry combined with an end-capture method for low-input amounts down to the single-cell level. As we have previously reported, SMART-Seq v4 technology produces high-quality cDNA libraries from individual cells that closely represent the original in vivo mRNAs. It is extremely sensitive, works with transcripts of different lengths, and has excellent gene body coverage across a wide range of GC content (see References for details). In order to reduce labor and cost, we have adapted this robust technology for 3' end capture, and here we present this approach and validate it by sequencing single K562 cells.
This approach allows each pool of cDNA from 12 single-cell reactions to be tagged by one of the 96 Illumina HT barcode combinations, enabling up to 1,152 separate cell reads per run. Researchers using this kit can confidently determine differentially expressed transcripts while decreasing the cost and time required for discovery. This method utilizes a modified oligo(dT) primer including an in-line index which serves as a cell barcode and a portion of the Illumina read primer 2 sequence in order to accommodate a pooled library generation protocol (Figure 1). The in-line index is placed between the transcript and the Illumina read primer 2 (RP2), and enables pooled cell samples to be demultiplexed after sequencing.