What you are doing ought to work..

However, at least one report suggests MTSEA biotin xx might label the 4SU better for your application than the HPDP.

https://www.ncbi.nlm.nih.gov/pubmed/26340425

Thank you for your answer. I reached out to the authors and they had said that MTSEA biotin XX does have higher efficient (which the literature states), but there is also higher total RNA contamination. However, if HPDP doesn't work this time around I will give it a shot.

How much input RNA are you putting into the pulldown? I have a colleague that did a similar experiment (10 min 4sU labelling, biotinylation and strepdavidin pulldown). They used 75 µg RNA input and got maybe ~ 100 ng RNA at the end (at best). So the output is pretty low in comparison to what you put in. 

Hi William, 

Thank you for your reply. I have had trouble getting high yields of RNA with high purity, so for the pull down I would have about 20ug total RNA, which now sounds way too low.  I start off with around 60ug after the initial extraction, but lose a lot after the biotin labeling with column/ethanol precipitation. I think I will need to pull more plates together when I do the RNA extraction to get higher yields. I am using four 10cm plates of MCF10A cells (~35x10^7 cells). Do you mind if I ask which cells your colleague was using? Thank you again, that is good information to have. 

Ah I'm not sure how much goes into the actual pulldown, I just know that 75 µg goes into the biotin labelling. So it could be that there could be a similar amount going into the pulldown, although they did not EtOH precipitate after - just purified over P-30 gel columns. The cells are E14 mouse ES cells - I think it was from two 10 cm plates but I would have to check the protocol again. 

Good morning,

I personally tried this protocol as well with a 10min pulse labelling on mES cells. However I am facing a serious problem with HPDP-biotin that precipitates during the biotinylation step. I tried both DMF and DMSO for stock preparation (at 1mg/ml), which is fine, but precipitation still occurs. Even if I add 40% (instead of 20%) solvent in the biotinylation mix, it still precipitates.

As a consequence, I obtain only a few ng of labeled RNAs which is not sufficient for RNA-seq...

Does anyone face also this problem? I am completely hopeless.

Thank you

Hi Paul,

I ended up doing a 10 minutes pulse as well for the 4SU. I did not have a precipitation of the 4SU solution in DMF though.

In the end I received only a few ngs of RNA, but if you use the

Ovation SoLo RNA-Seq System for RNA seq prep it can be done. My input was between 150-200ug of total RNA for the pull down, so if you are able to increase this amount I think this may help. Best of luck!

Carly Harro

Hi Carly,

I have been doing the 4SU-seq recently, and I met the same problems as you did. I input ~ 150 ug RNA and only get 100~200 ng new transcribed RNA yield even with extended to 1 hour labeling.

Did you figure out the problem? Could you give some advice?

Thank you.

Hey Jiale,

I label my E14 mESC with 4sU for 10 or 15 minutes. I extract total RNA and biotinylate 100 ug (HPDP-biotin). After precipitation I end up with about 80-90 ug of biotinylated RNA. When I input this onto the streptavidin beads I end up with about 400-500 ng of newly transcribed RNA. The important thing is to always perform the protocol, with a ctrl of RNA extracted from cells not labelled with 4sU... this is your baseline, you should always compare the enrichment of your 4sU labelled samples to your non labelled control... Few ng of yield after 1 hour of laballing seems quite low though... what concentration of 4sU are you using? What beads are you using? You can increase the concentration of beads you use, just make sure this does not increase the amount of RNA you pull down from your background unlabelled control. Hope this helps

Hi Jiale,

I ended up doing a similar protocol to Andriano.

I lowered my 4SU to 500uM, but increased the labeling time to 10 minutes. I had much lower yield of purified 4SU labeled RNA, but it still worked for sequencing (like ng-pg range). I just had to use an Illumina kit that worked for the range.

I would start with a lot of cells. I had adherent cells, so I ended pulling multiple plates together to have enough total RNA at the start of the protocol.

I would not use 1hr labeling as this has been reported in the literature to have problems.

Products:

4-thiourdine (Sigma-Alrich)

Dynabeads Streptavidin

Good luck!

Adriano Biasini and Carly Harro ,Thank you so much for your help! I really appreciate all your explanation.

I troubleshooted my problem out. I used uMacs beads and 200 uM 4SU / 10 mins labeling which gave me the same yield as Adriano did.

Thank you again!

Hi! Did anyone of you use this method to calculate mRNA half life?

Hi Ivana,

yes we did, we used INSPEcT pipeline (De Pretis et al. 2015) on RNA Seq performed on total and newly transcribed (4sU label led) RNA to infer transcriptome wide data. If you would like to test by qPCR (quality control before RNA Seq) you can use the method presented in Doelken et al.2008, that is how we optimized the protocol and confirmed what we were enriching for in the pull down step really was newly transcribed RNA