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Old 07-13-2016, 09:10 AM   #1
Location: Boston

Join Date: Aug 2015
Posts: 38
Default IDT xGen kit on RNAseq libraries?

I'm looking into using the xGen lockdown kit to select for a panel of specific genes from our RNA seq samples. These are low input samples, so my normal workflow is doing about 13 cycles of preamp, then pool and SPRI clean. Then I take that into Nextera, which is then amplified again with the Nextera handles for 12 cycles. This usually produced me with sufficient DNA for a NextSeq run (1-4nM, maybe about 10-20uL total).

I'm looking into doing a gene enrichment with the xGEN kit after the preamp. but the recommended input of the kit seems really high. It asks for 500ng of material, while my preamp material is usually around 1-2ng/uL (15uL total). Does anybody have any idea whether the kit would still work with much lower input? Or is there a different kit that would be more suited for this purpose? (the libraries are already barcoded. so no additional barcoding is necessary.)
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Old 07-18-2016, 10:41 AM   #2
Epigenomics NGS Beast
Location: New Jersey

Join Date: Oct 2010
Posts: 17

I've tried the kit with lower input from stranded RNA library preps and it seems to be fine at low coverage (~40X). i'd probably try to stay above 150-200ng for the hybs if you're trying to cut down on input. If duplication rates become an issue you might want to increase input, but it will depend on how deep you want to sequence and on the complexity of your library, pre-cap.
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