Dahlquist:RNA-seq Protocol: Difference between revisions
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== Poly(A) Enrichment == | |||
# Aliquot 10 μL of oligo(dT) beads (NEB). | |||
#* NOTE – this amount of the NEB beads suffices for quantities of total RNA below 10 µg. We have not explored larger quantities of RNA, as this seems exorbitant. Also, we have used as little as 100 ng of total RNA with good results. Lower quantities should work as well, but one should keep in mind that non-specific binding of RNA to beads may reduce the enrichment of poly(A) RNA. Also, it is probable that the starting amounts of poly(A) RNA may fall below the affinities of RT for template DNA if one starts with less than 100 ng of total RNA. This needs to be accounted for in the following steps (specifically, step 2), possibly by extending RT times and perhaps periodically adding more enzyme. | |||
# Wash the beads twice with 100µL of Binding Buffer (20mM Tris-HCl pH 7.5, 1.0M LiCl and 2mM EDTA), and remove the supernatant. | |||
# Resuspend the beads in 50 µl of Binding Buffer. | |||
== Useful Links == | == Useful Links == | ||
[http://core-genomics.blogspot.com/2012/04/how-do-spri-beads-work.html ''How do SPRI beads work?''] by James@cancer. | [http://core-genomics.blogspot.com/2012/04/how-do-spri-beads-work.html ''How do SPRI beads work?''] by James@cancer. | ||
Revision as of 10:23, 28 June 2016
Poly(A) Enrichment
- Aliquot 10 μL of oligo(dT) beads (NEB).
- NOTE – this amount of the NEB beads suffices for quantities of total RNA below 10 µg. We have not explored larger quantities of RNA, as this seems exorbitant. Also, we have used as little as 100 ng of total RNA with good results. Lower quantities should work as well, but one should keep in mind that non-specific binding of RNA to beads may reduce the enrichment of poly(A) RNA. Also, it is probable that the starting amounts of poly(A) RNA may fall below the affinities of RT for template DNA if one starts with less than 100 ng of total RNA. This needs to be accounted for in the following steps (specifically, step 2), possibly by extending RT times and perhaps periodically adding more enzyme.
- Wash the beads twice with 100µL of Binding Buffer (20mM Tris-HCl pH 7.5, 1.0M LiCl and 2mM EDTA), and remove the supernatant.
- Resuspend the beads in 50 µl of Binding Buffer.
Useful Links
How do SPRI beads work? by James@cancer.
