ARTSeq Ribosome Profiling

[SallyH]

Has anyone used the ARTSeq Ribosome Profiling kit from Epicentre? I am finding one step quite confusing and don't appear to be getting anywhere with Epicentre tech support.

I would appreciate it if anyone who has used the kit could answer a question for me please!

[SallyH]

I am going through the ARTSeq Ribosome profiling kit to clearly understand
it before I start work.

Section 3A (pg 10 of the ARTSeq kit) details the removal of mammalian rRNA
using Ribo-Zero Magnetic kit.

3A.2 (pg 10 of the ARTSeq kit) states to follow the Ribo-Zero kit
procedure, except omit step 3.C.3. 3A.3 (again of the ARTSeq kit) then
states purify Ribo-Zero treated samples using the Zymo Research RNA Clean
and Concentrator kit.

I therefore have two questions:

1. Do you follow the Ribo-Zero Magnetic kit right until the end and purify
the rRNA depleted sample (using the method of your choice Section 3.D) and
then purify again using the Zymo Research RNA Clean and Concentrator kit
(as detailed in 3A.3 of the ARTSeq kit). Or do you omit step 3.D of the
Ribo-Zero kit procedure too and just proceed to the Zymo Research RNA
Clean and Concentrator kit. I'm sorry, but I am just failing to
understand why the samples appear to need purifying twice; once at the end of the
Ribo-Zero procedure and then again with the Zymo Research RNA Clean and
Concentrator kit.
2. What is the rational for having two modified Zymo Research RNA Clean
and Concentrator kit protocols depending on the sample (total RNA or
ribosome protected RNA) 3A.3 of the ARTSeq protocol?

[scooter]

For your first question, it is my understanding that following the rRNA depletion with Ribozero the material should be purified using the Zymo kit only. So there is not two different purification steps. They also supply a slightly altered zymo protocol that mostly just adds extra ethanol to the sample before passing over the column. This is presumably because the RNA fragments are so small that the extra ethanol is added to insure you can capture the fragments efficiently.

For your second question. I think the two different zymo protocols suggested are because the two samples (Total vs. the footprints) are very different samples. The footprints are very small RNA fragments generated by nuclease treatment, but the total RNA is larger transcripts. for the Total RNA samples, using the Zymo protocol for the larger RNAs (the >200 nt protocol) makes more sense because it will remove small RNAs (like tRNA) that are not desired, yet retain the desired larger RNAs. Since tRNA and other non-coding RNAs can be very abundant, it would make sense to remove them from the sample before library prep.