Question: What is the difference between a Single Cell 3' and Single Cell 5’ Gene Expression library?
Answer: The protocols are similar but different ends of polyadenylated transcripts are captured in the final 10x 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 in the 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).
Both 3' and 5' Gene Expression libraries have the same recommended sequencing configuration (Read 1: 26 cycles, i7 index: 8 cycles, i5 index: 0 cycles, Read 2: 98 cycles) and sequencing depth (50,000 raw reads per cell).
A schematic diagram comparing the final library construct for the two assay schemes is illustrated below. Additional information can be found in the Assay Scheme and Configuration Technical Notes for each solution in the corresponding link:
Single Cell v2 3' Gene Expression Library:
Single Cell 5' Gene Expression Library:
Products: Single Cell 3', VDJ