Dahlquist:RNA-seq Protocol: Difference between revisions
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# 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. | # 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. | # Resuspend the beads in 50 µl of Binding Buffer. | ||
# Bring RNA to 50 µL with RNAse-free water. Heat to 65°C, cool on ice, then add to the washed beads. Incubate at room temperature for 5 min. | |||
# Collect beads using the magnetic stand, discard supernatant. | |||
# Wash the beads twice with 100µL of Washing Buffer B (10mM Tris-HCl PH 7.5, 0.15M LiCl, 1mM EDTA). | |||
# Remove the supernatant from the beads, add 15 µL of 10mM Tris-HCl (this depends on how much RNA is used, and how many samples the polyA RNA is going to be used for) and heat the beads at 800C for 3 minutes to elute mRNA. | |||
# Remove beads with the magnetic stand, save the supernatant. | |||
#* NOTE: it may be advisable to repeat this, to remove as much rRNA as possible. However, this is still an open issue, since organellar and stable RNAs are known to be polyadenylated, which means repeated rounds of poly(A) enrichment yielding reduced returns. | |||
#* Also, since we use NEB oligo-dT beads, it should be OK to follow NEB’s protocol for poly(A) enrichment instead of the one I described in the preceding. NEB and other companies sells kits as well as just the beads, and some may prefer to use the ready-made solutions and protocol. | |||
== 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 10:25, 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.
- Bring RNA to 50 µL with RNAse-free water. Heat to 65°C, cool on ice, then add to the washed beads. Incubate at room temperature for 5 min.
- Collect beads using the magnetic stand, discard supernatant.
- Wash the beads twice with 100µL of Washing Buffer B (10mM Tris-HCl PH 7.5, 0.15M LiCl, 1mM EDTA).
- Remove the supernatant from the beads, add 15 µL of 10mM Tris-HCl (this depends on how much RNA is used, and how many samples the polyA RNA is going to be used for) and heat the beads at 800C for 3 minutes to elute mRNA.
- Remove beads with the magnetic stand, save the supernatant.
- NOTE: it may be advisable to repeat this, to remove as much rRNA as possible. However, this is still an open issue, since organellar and stable RNAs are known to be polyadenylated, which means repeated rounds of poly(A) enrichment yielding reduced returns.
- Also, since we use NEB oligo-dT beads, it should be OK to follow NEB’s protocol for poly(A) enrichment instead of the one I described in the preceding. NEB and other companies sells kits as well as just the beads, and some may prefer to use the ready-made solutions and protocol.
Useful Links
How do SPRI beads work? by James@cancer.
