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Old 02-08-2017, 03:55 AM   #1
sahilgupta
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Location: Frankfurt

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Default RNA seq Library Purification

Dear All!

Hello, I am working on RNA sequencing NextSeq500 TrueSeq mRNA LT protocol. I have residual adapters after AmPure purification in 2/8 samples. Should I gel extract my these two or all samples or purify with AmPure beads again?

I am wondering, if I might loose sample with AmPure purification. I used .85X beads ratio already.


Any Recommendations? (Adapter dimer starts at 110bp and my library at 200bp)

Regards
Sahil Gupta
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Old 02-08-2017, 01:20 PM   #2
nucacidhunter
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Any purification method will cause loss of DNA with beads having relatively less loss than gel extraction. 0.85x bead should have cleaned the dimers if the protocol was followed without modification using calibrated pipettes. Two options:
1- clean all samples if they are from the same experiment to reduce introducing another variable
2- sequence samples with dimers deeper to compensate for the dimer reads that will be cleaned from data
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Old 02-12-2017, 11:59 PM   #3
sahilgupta
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Thank you! It works with .8X beads purification!!!
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