Question: Why are the Universal Blockers added after the SPRIselect step in Demonstrated Protocol CG000249?
Answer: In the SPRIselect Library Concentration Protocol (CG000249), the Universal Blockers are added only after the SPRIselect step, given that their fragment size is too short to be retained in a 1.8X SPRIselect cleanup.
Universal Blockers are short DNA oligos that bind to the adapter sequences of 10x Genomics library fragments to reduce off-target hybridization during the Targeted Gene Expression assay.
In the Demonstrated Protocol for SPRIselect Library Concentration (CG000249), SPRIselect beads are added to the libraries at a 1.8X ratio. At this 1.8X ratio, DNA fragments greater than ~100bp will bind to the beads and will be retained, while shorter fragments will remain in the supernatant and will subsequently be discarded. If the Universal Blockers were added prior to the SPRIselect beads, they would be discarded in the supernatant.
Note: The Targeted Gene Expression assay has been discontinued. For alternative options, please see: What options are there for performing a targeted enrichment for my gene expression libraries?
Products: Targeted Gene Expression