Question: If full-length mRNA is transcribed, how is the assay biased to 3' or 5'?
Answer: Only the 3' or 5' portion of the transcript is retained in the steps after enzymatic fragmentation. The fragmentation produces molecules of varying sizes. After end-repair, A-tailing, and ligation of Read 2, the fragmented molecules are amplified with primers that only recognize fragments that contain the Read 1 sequence, which is supplied on the gel bead oligo (along with the 10x barcode, UMI, and transcript fragment) and Read 2 sequence.
This schematic illustrates the library construction for Single Cell 3' libraries:
For the Single Cell V(D)J library construction steps, please see the User Guide.
Products: Single Cell Gene Expression, Single Cell Immune Profiling