Question: What is the difference between Single Cell 3' and Single Cell 5’ Gene Expression libraries?
Answer: The two assays are similar but capture different ends of the polyadenylated transcript in the final library. Both solutions use polydT primer for reverse transcription, although in the 3' assay the polydT sequence is located on the gel bead oligo, while in the 5' assay the polydT is supplied as an RT primer. A template switching oligo (TSO) is used in both workflows to reverse transcribe the full-length transcript.
After amplifying the cDNA, molecules are randomly fragmented under conditions that favor 300-400 bp length fragments. Downstream of fragmentation, only transcripts containing both (1) a 10x Barcode AND (2) an Illumina Read 2 adaptor, which is ligated on to the cDNA after fragmentation, will be amplified during the Sample Index PCR. This results in final 10x libraries that either represent the 3' end of the transcript (as the 10x Barcode is adjacent to the polyA tail on the 3' end of the transcript) or the 5' end of the transcript (as the the 10x Barcode is adjacent to the TSO and the 5' end of the transcript).
A schematic diagram comparing the final library construct for the two assay schemes is illustrated below. Note that the library construct for different Single Cell 3' reagent chemistries (v2 and v3) is similar, but the UMI is longer for v3. The required number of sequencing cycles is also different.
Single Cell 3' v3 Gene Expression Library:
Products: Single Cell 3', VDJ