Question: Can I re-amplify Single Cell libraries if my yield is too low for sequencing?
Answer: If the final 10x Single Cell 3' or 5' library yield is too low for sequencing, it is possible to re-amplify the final library with generic Illumina TruSeq primers.
We recommend using 7 additional PCR cycles for amplification. The oligo sequences of the Illumina TruSeq primers are:
P5: 5' AAT GAT ACG GCG ACC ACC GA 3'
P7: 5' CAA GCA GAA GAC GGC ATA CGA 3'
The protocol for the PCR re-amplification is as follows:
- 0.5uM P5 and 0.5uM P7 primers
- 50 uL 2x Amplification Master Mix (any generic PCR amplification master mix can be used)
- 20 uL 10x Single Cell 3' or 5' Library
- Water (to bring up final reaction volume to 100 ul)
|5||Go to Step 2, 6X (for 7 cycles total)|
After amplification, perform a 1x SPRI cleanup to remove primers.
We have not observed that re-amplification introduces any bias in the final sequencing data assuming that the fragments are flanked by P5 and P7 sequences.
Products: Single Cell 3', VDJ