Question: Are the random nucleotides (Ns) in the feature barcode oligo conjugation required?
Answer: Our testing in house utilized the oligo structure published in our Demonstrated Protocol: https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstrated-protocol-cell-surface-protein-labeling-for-single-cell-rna-sequencing-protocols
While excluding the random bases will still enable the successful production of cell surface protein libraries, we do still recommend keeping the N's as they:
- Help to increase the length of the final library construct (making it > 200bp)
- Add stretches of diversity, which is beneficial if the libraries are sequenced without pooling with their corresponding gene expression libraries
- Improve sequencing quality by giving the sequencers random bases to calibrate base-calling before mono-template issues with the feature barcode
Note: When comparing the quality differences in Antibody Application metrics (contained in the web summary output), the final performance with or without Ns was similar. Also, if the N’s are omitted, the feature reference file will need to be adjusted to take this into account.
Please refer to our support website for information on Feature Barcoding analysis:
Single Cell 3' v3:
Single Cell VDJ:
https://support.10xgenomics.com/single-cell-vdj/software/vdj-and-gene-expression/latest/overview
Products: Single Cell Gene Expression, Single Cell Immune Profiling