Hi Tony,

We did two cycles of poly(A)+ extraction using magnetic oligo(dT) beads included in the library construction kit for mRNA sequencing (by Illumina). Removal of rRNA should theoretically be better than poly(A)+ extraction. For example, we obtained far lower histone reads than expected from northern data in Acytostelium. I have no experience with Ribominus but would vote for yeast. However, it could depend on the region of rRNA they used as a target of oligo. Maybe people in the company will help if you send the rRNA sequence in Dicty?

- Hideko

From: DICTY [mailto:[log in to unmask]] On Behalf Of Richard Sucgang, PhD
Sent: Thursday, September 02, 2010 1:02 AM
To: [log in to unmask]
Subject: Re: [DICTY - 402] RNA sequencing

I believe that the standard polyA enrichment for mRNA should work If you are primarily interested in that fraction of the RNA pool, but otherwise I would recommend the yeast kit. There is sufficient cross hybridization between the yeast and the Dicty rRNA that yeast probes were initially used to characterize them early on.  

On Sep 1, 2010, at 10:06 AM, Anthony Kowal <[log in to unmask]> wrote:
Hi Everyone,

We are about to send some samples for RNA sequencing, and I had a question about initial sample preparation that I hope someone can answer.  

The samples that I am submitting are from a total RNA isolation from Dictyostelium, and the RNA sequencing will be performed using the SOLiD system.  I understand the overall scheme about how the RNA sequencing works, but my question regards the removal of ribosomal RNA species from my samples.  Our core facility here uses a step using "Ribominus", and they are asking about which Ribominus kit they should use - one for human and mouse or one for yeast.  I am not sure which one would be best, or if people in the Dictyostelium community even do this step.

I would deeply appreciate any suggestions and information that could be provided.  

Thank you very much.

Sincerely,

Tony

-- 
Tony Kowal
Graduate Student
Chisholm Laboratory

Northwestern University
303 E. Superior 
Lurie Building
Room 7-250
Chicago, IL 60611

Lab: 312 503 4169
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