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Forum: Illumina/Solexa 03-13-2018, 02:15 AM
Replies: 1
Views: 647
Posted By huguesparri
We used the Truseq kit succesfully on Bacteria. 2...

We used the Truseq kit succesfully on Bacteria. 2 things are essential:
- DNAse treatment of your RNA.
- Good depletion efficiency for the ribosomal RNA. Ribozero kit are usually doing well.
Forum: Illumina/Solexa 02-20-2018, 05:02 AM
Replies: 0
Views: 822
Posted By huguesparri
All good things must come to an end...

Only five years to go :'(
Forum: Sample Prep / Library Generation 11-03-2017, 01:59 AM
Replies: 2
Views: 960
Posted By huguesparri
Hello, Did you try the Agencourt SPRIPlate 96...

Hello,
Did you try the Agencourt SPRIPlate 96 (ref. A29164 in the beckman Coulter catalog)?
We use it in our lab and we are rather satisfied with it.
regards,

Hugues
Forum: Sample Prep / Library Generation 10-09-2017, 05:27 AM
Replies: 7
Views: 3,568
Posted By huguesparri
The kit includes so-called "SPB" (Sample...

The kit includes so-called "SPB" (Sample purification Beads) so you don't need AMpure XP anymore (except for an initial hypothetic sample purification).
It might not justify completely the higher...
Forum: General 09-27-2017, 05:55 AM
Replies: 1
Views: 970
Posted By huguesparri
Yes we did: we are sealing the plates manually....

Yes we did: we are sealing the plates manually. So far, it's the easiest and surest way we found to prevent evaporation.
Sorry if it's not the answer you were hoping for :s
Forum: Illumina/Solexa 09-19-2017, 05:44 AM
Replies: 5
Views: 1,142
Posted By huguesparri
Thanks for the tip. We'll keep it in mind next...

Thanks for the tip.
We'll keep it in mind next time we plan some RRBS experiment :)
Forum: Illumina/Solexa 09-14-2017, 06:35 AM
Replies: 5
Views: 1,142
Posted By huguesparri
We have tested the Diagenode kit with success (24...

We have tested the Diagenode kit with success (24 reactions).
For me the biggest issue is that the kit do not provide a lot of reagents in excess.
And as you have to pool you samples before the...
Forum: Sample Prep / Library Generation 09-14-2017, 06:28 AM
Replies: 1
Views: 683
Posted By huguesparri
Hello, In my opinion, the most sensitive...

Hello,

In my opinion, the most sensitive reagent in the kit is the ligation mix.
But it's hard to say if it suffered from standing at RT without testing it.
if you have any doubt, you can try to...
Forum: Sample Prep / Library Generation 07-19-2017, 11:34 PM
Replies: 2
Views: 674
Posted By huguesparri
Hello, This kind of protocol is typical of...

Hello,

This kind of protocol is typical of smallRNAseq.
It uses a truncated T4 RNA ligase 2 and a modified 3' adapter.
Trunc. T4 RNA ligase is unable to adenylate the 5' end of its substrate so...
Forum: Illumina/Solexa 07-19-2017, 04:42 AM
Replies: 6
Views: 1,097
Posted By huguesparri
I agree with both of you regarding the quality of...

I agree with both of you regarding the quality of the machine itself. As I said, we have one for 3 years now and we are completely satisfied with it (set aside the price of the reagents).
But as...
Forum: Illumina/Solexa 07-18-2017, 02:02 AM
Replies: 6
Views: 1,097
Posted By huguesparri
You bought a HS2500? I thought the machine was...

You bought a HS2500? I thought the machine was being discontinued by Illumina.
Don't get me wrong: the machine itself is awesome, very flexible with high quality runs. We are using one for more than...
Forum: Sample Prep / Library Generation 07-10-2017, 04:46 AM
Replies: 5
Views: 1,351
Posted By huguesparri
Just be aware that in drosophila, 2S RNA is...

Just be aware that in drosophila, 2S RNA is abundant and 30nt long.
You may (will?) need a specific protocol to build smallRNAseq libraries from drosophila RNA.
Forum: Sample Prep / Library Generation 06-15-2017, 12:02 AM
Replies: 1
Views: 646
Posted By huguesparri
Hello, Did you only use the nanodrop to...

Hello,

Did you only use the nanodrop to quantify your RNA samples or did you also perform some integrity checking on bioanalyzer?
Your concentration is pretty low and your nanodrop ratios are not...
Forum: RNA Sequencing 03-17-2017, 02:51 AM
Replies: 7
Views: 1,604
Posted By huguesparri
I advise you to build all the libraries you're...

I advise you to build all the libraries you're going to compare at the same time to reduce technical biases.
Are you using a kit or a home made protocole?
Forum: Sample Prep / Library Generation 02-10-2017, 06:36 AM
Replies: 8
Views: 1,343
Posted By huguesparri
Maybe I missed something but you said that you...

Maybe I missed something but you said that you have "library prep issues".
Could you tell us what kind of protocol did you use to build your libraries?
Forum: Sample Prep / Library Generation 02-07-2017, 07:48 AM
Replies: 0
Views: 558
Posted By huguesparri
How to interprete Stacks partial results?

Hi,
I'm posting here but I'm not sure I'm in the correct section of the forum. If so, I apologize in advance.

So, we are performing RADseq experiments.
DNA comes from differents organisms and...
Forum: Sample Prep / Library Generation 02-06-2017, 04:43 AM
Replies: 4
Views: 1,636
Posted By huguesparri
You can take a look at this post: ...

You can take a look at this post:
http://seqanswers.com/forums/showthread.php?t=9967
It might be helpfull.
Forum: Sample Prep / Library Generation 01-19-2017, 02:34 AM
Replies: 10
Views: 2,115
Posted By huguesparri
It's only an issue if you're size selecting...

It's only an issue if you're size selecting before the PCR. At least, that's what we figured out from our experiments.
If you perform the PCR before the size selection, even if you loose material...
Forum: Sample Prep / Library Generation 01-18-2017, 07:40 AM
Replies: 1
Views: 907
Posted By huguesparri
Judging by the picture you joined to your...

Judging by the picture you joined to your message, they are added during the PCR step.
Indexing during PCR is rather common ine Illumina's protocols especially when for amplicons and when using...
Forum: Sample Prep / Library Generation 01-18-2017, 07:27 AM
Replies: 10
Views: 2,115
Posted By huguesparri
As sonication is performed before Immuno...

As sonication is performed before Immuno precipitation, you should be able to see your sonication profile on a bioanalyzer, especially when starting from 50 millions cells.
I advise to quantify you...
Forum: Sample Prep / Library Generation 11-17-2016, 05:19 AM
Replies: 4
Views: 1,286
Posted By huguesparri
Usually we use a Bioruptor because it's located...

Usually we use a Bioruptor because it's located in our building.
But we also have access to a S220 in the university. When we used it, the parameters were as follow:
Volume: 130l.
DNA: 1 g....
Forum: Sample Prep / Library Generation 11-16-2016, 08:33 AM
Replies: 4
Views: 1,286
Posted By huguesparri
Hello, Usually, we perform a AMPure XP...

Hello,

Usually, we perform a AMPure XP purification step after the P1 ligation and we do not face any issue with the sonication. Maybe you should give it a try...

H.
Forum: Sample Prep / Library Generation 10-17-2016, 07:38 AM
Replies: 2
Views: 1,161
Posted By huguesparri
Hi Irene, First of all, I don't have experiment...

Hi Irene,
First of all, I don't have experiment with the nextseq system. We have a Hiseq2500 but I suppose the issue and solutions are the same.
What we usually do when our library concentration is...
Forum: Illumina/Solexa 10-05-2016, 06:11 AM
Replies: 16
Views: 4,440
Posted By huguesparri
For the RNA part, we use the truseq RNA stranded...

For the RNA part, we use the truseq RNA stranded sample prep kit and protocol.
For the RAD part, we use the Baird protocol. We just replaced the column purification steps with AMPure XP purification...
Forum: Illumina/Solexa 10-05-2016, 05:15 AM
Replies: 16
Views: 4,440
Posted By huguesparri
We use a Tecan evo to build RNAseq libraries and...

We use a Tecan evo to build RNAseq libraries and RADseq libraries and we are happy with it.
We often process 96 samples at the same time.

For the RAseq part, we ends up with less material than...
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