In fact you must not remove the ribosomal RNA from your sample. That is because, that is the RNA which you want.

Of course you need to remove nascent robosomal RNA ! Usually, ribozero from epicenter (MRZH116) is used. Then you can find different protocol for library preparation in several articles. For example, you can find this protocol after BrU Run on nascent RNA in this link

http://www.sciencemag.org.gate1.inist.fr/content/suppl/2008/12/04/1162228.DC1/Core.SOM.pdf

wich is the supp material and method from coreJ and Lis JT, Science 2008, or in this pub from P Cramer's lab (attached file) in the supp data for 4tUseq.

As 4tU seems to me to be much closer to the technique developped in the clik-it kit, you might choose their protocol. 

I hope I helped

Cheers

Anne

It depends on the protocol you want to use for RNA-Seq library preparation. After the click-it you will end-up with purified 'nascent' RNA which still needs to be processed to generate a RNA library suitable for Next-Gen sequencing. During this process you typically use a poly-A selection, then chemical fragmentation and finally random hexamer based cDNA synthesis. After the click-it the RNA will be purified already but poly-A tails will be still available for a second round of purification/selection, so imho the point is: are you planning to use the poly-A selection? if so, removal of rRNA is not strictly necessary, otherwise it is.

Thanks for the help Anne and Rocco. To answer your question Rocco - I dont want polyA selection but rather a view of the nascent transcriptome as it is with non-coding unprocessed transcripts. I guess then removal of ribosomal RNA is best (since I dont care about that fraction). Thanks again.

Hi, I am planing to use the Click-iT Nascent RNA Capture kit (Invitrogen). Have you tried? Do you think it can be used for sequencing? I didn't find any paper use this kit to do sequencing, many of them just performed RT-qPCR. Thank you.

Dear all,

I am also facing this technology for the first time and I would like to know how it went for you! Suggestions/advice?

I am wandering if I should do riboselection and then ask the facility to process everything from the beads (doing RNA fragmentations directly on the beads and so on), or better provide them with the cDNA directly!

Thanks a lot and regards to all,

Luisa

Short answer: Yes, you need to remove it.

Long answer:

This paper that came out recently used nascent RNAseq: Tong et al 2016, Cell.

In their experimental procedures state that:

Total RNA and chromatin-associated RNA were prepared as described (Bhatt et al., 2012). Strand-specific libraries were generated from 60 ng chromatin RNA or 400 ng total RNA using the TruSeq RNA Sample Preparation Kit v2 (Illumina) and the dUTP second strand cDNA method (Levin et al., 2010). cDNA libraries were single-end sequenced (50bp) on an Illumina HiSeq 2000.

If you go to Bhatt et al 2012 experimental procedures:

Subcellular fractions were prepared as described (Pandya-Jones and Black, 2009), with minor changes. The cell lysis buffer contained 0.15% NP-40, and the sucrose cushion did not contain detergent. Fraction purity was confirmed by immunoblot analysis of SNRP70, β-tubulin (Sigma), and histone H3 (Abcam). Cytoplasmic and nucleoplasmic RNA was purified by using QIAGEN RNeasy columns. Chromatin RNA was isolated by using TRI-reagent (MRC), followed by further purification with RNeasy columns. All samples were eluted into 100 ul RNase-free water. RNA (10 ug) from each fraction was depleted of ribosomal RNA (rRNA) by using the Mouse/Human Ribominus Kit (Invitrogen). Whole-cell RNA was purified using TRI-reagent and RNeasy columns. Polyadenylated RNA was purified by using the MicroPoly(A) Purist Kit (Ambion).

I hope this helps

Miguel