Question: Can I amplify my final library if my yield is too low?
Answer: In theory, it is possible to re-amplify the library, although you will not have enough 10x-supplied kit reagents to do so (and have enough for subsequent experiments). With that being said, we've successfully sequenced libraries with cDNA yields as low as 1-2 ng.
You can 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.
If the low library yield stems from low cell input or degraded mRNA, then re-amplifying the library may not increase the number of unique transcripts detected.
Products: Single Cell 3', Single Cell VDJ