Adapter titration in TruSeq stranded mRNA sample preparation

chicken · 11-11-2016, 04:19 AM

Hi,
I am doing transcriptome analysis and prepared some RNA libraries from total RNA. Allways using 500ng of total RNA. I always got the problem that I got a lot of adapter dimers in the end. I check it by loading sample on an agarose gel. I suspect that I somehow have to titarate the adapter but I do not know how since there are no suggestions in the prepration guide and I do not know the concetration of the adapter-mix is. Have anybody done a titration before with that certain preparation kit?? I would be really happy about a little hint.

Thanks in advance.

Used Kit: TruSeq stranded mRNA sample preparation by Illumina

NGSeqer · 11-11-2016, 01:18 PM

Hi Chicken,

The AMPure bead cleanup after adapter ligation should remove most adapter dimer. Things that can affect bead cleans up are: using incorrect bead to sample ratio, expired beads, and not using freshly made EtOH.

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11-11-2016, 04:19 AM	#1
chicken Junior Member Location: Europe Join Date: Sep 2016 Posts: 3	Adapter titration in TruSeq stranded mRNA sample preparation Hi, I am doing transcriptome analysis and prepared some RNA libraries from total RNA. Allways using 500ng of total RNA. I always got the problem that I got a lot of adapter dimers in the end. I check it by loading sample on an agarose gel. I suspect that I somehow have to titarate the adapter but I do not know how since there are no suggestions in the prepration guide and I do not know the concetration of the adapter-mix is. Have anybody done a titration before with that certain preparation kit?? I would be really happy about a little hint. Thanks in advance. Used Kit: TruSeq stranded mRNA sample preparation by Illumina

11-11-2016, 01:18 PM	#2
NGSeqer Junior Member Location: IN Join Date: Sep 2016 Posts: 1	Hi Chicken, The AMPure bead cleanup after adapter ligation should remove most adapter dimer. Things that can affect bead cleans up are: using incorrect bead to sample ratio, expired beads, and not using freshly made EtOH.