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Forum: Sample Prep / Library Generation 11-14-2012, 03:46 PM
Replies: 0
Views: 1,458
Posted By CC_seqanswers
ion torrent small RNA library size issue

I have been running my RNA samples through Ion torrent small RNA protocol v2(gel free) a few times but haven't got good luck with it.My library size is always 10 bp smaller than expected (76bpvs...
Forum: Sample Prep / Library Generation 06-05-2012, 07:29 AM
Replies: 4
Views: 2,667
Posted By CC_seqanswers
Thanks for your help. Pls see replies below.

Thanks for your help. Pls see replies below.
Forum: Sample Prep / Library Generation 06-04-2012, 10:31 AM
Replies: 4
Views: 2,667
Posted By CC_seqanswers
Thank you! We have been quite succesful with NEB...

Thank you! We have been quite succesful with NEB RNA fragmetation for pair end sequencing too.

It's just this time we are using Ambion kits and we need to use RNaseIII.

:(
Forum: RNA Sequencing 06-04-2012, 09:59 AM
Replies: 8
Views: 4,566
Posted By CC_seqanswers
It's normal. The two groups might just have...

It's normal.

The two groups might just have different number of libraries running on a flowcell or library normalization was off before emPCR. The number of reads your sample has depends on the...
Forum: Sample Prep / Library Generation 06-04-2012, 09:50 AM
Replies: 4
Views: 2,667
Posted By CC_seqanswers
RNA fragmentation using RNAse III: size issue

I am trying to make RNA libraries for pair end sequencing(2x100). However I failed to get the RNA fragment size I need by using RNase III fragmentation. The average size of fragmented...
Forum: Sample Prep / Library Generation 05-10-2012, 07:58 PM
Replies: 23
Views: 8,684
Posted By CC_seqanswers
The sequencing primers in Truseq sequencing kit...

The sequencing primers in Truseq sequencing kit are a cocktail of all kinds of sequencing primers: those for ILMN pair end libraries, for truseq libraries and even for v1.5 sRNA libraries.
Forum: Sample Prep / Library Generation 05-10-2012, 07:55 PM
Replies: 23
Views: 8,684
Posted By CC_seqanswers
I am confused by this information. DSN is...

I am confused by this information. DSN is supposed to take place on either ligated cDNA or amplified libraries. How does it have anything to do with polyA selection?
Forum: Sample Prep / Library Generation 04-18-2012, 08:53 PM
Replies: 23
Views: 8,684
Posted By CC_seqanswers
I have never done DSN on any TruSeq libraries but...

I have never done DSN on any TruSeq libraries but have been successful with older version of illumina RNA seq. I made libraries (purified PCR product) and performed DSN and amplified at the end....
Forum: Sample Prep / Library Generation 04-18-2012, 08:09 PM
Replies: 3
Views: 3,609
Posted By CC_seqanswers
Could you elaborate a bit on the experience with...

Could you elaborate a bit on the experience with Turbo? It worked great for you in terms of very low gDNA reads in the data? good yield of RNA? Have you compared the data between treatment and...
Forum: Illumina/Solexa 02-20-2012, 08:37 PM
Replies: 22
Views: 11,561
Posted By CC_seqanswers
Could you elaborate a bit on how the results with...

Could you elaborate a bit on how the results with small RNA are better than with total RNA?Thanks.
Forum: Sample Prep / Library Generation 10-18-2011, 08:01 PM
Replies: 22
Views: 11,921
Posted By CC_seqanswers
Is it because ethanol precipitation tends to...

Is it because ethanol precipitation tends to loose small RNA while Zymo promises to keep everything above 17?

...but at the end of the day, most sequencing protocol will remove small fragments...
Forum: Sample Prep / Library Generation 10-18-2011, 07:30 PM
Replies: 28
Views: 22,516
Posted By CC_seqanswers
It's true that Ovation method is like whole...

It's true that Ovation method is like whole transcriptome sequencing protocol, however, I still don't quite get that >50% reads mapped to intergenic area.

Would it be possible a slight amount of...
Forum: Sample Prep / Library Generation 10-12-2011, 08:54 PM
Replies: 28
Views: 22,516
Posted By CC_seqanswers
Hi, Bluescript, Thanks for your post. We...

Hi, Bluescript,

Thanks for your post. We have had good experience with Ovation kit too.

One thing I don't quite understand is why more than half of reads mapped to intergenic regions with...
Forum: Sample Prep / Library Generation 08-01-2011, 08:48 AM
Replies: 8
Views: 3,410
Posted By CC_seqanswers
May I ask what sizing technique in your case?

May I ask what sizing technique in your case?
Forum: Sample Prep / Library Generation 08-01-2011, 07:10 AM
Replies: 12
Views: 4,992
Posted By CC_seqanswers
Very impressive information.:) Thanks again! ...

Very impressive information.:) Thanks again!

Seems the adaptor/ligation design is quite much like SOLiD libraries.Hope 454 will adopt A-tail/Y shape adaptors for PE libraries soon.

We have been...
Forum: Sample Prep / Library Generation 07-28-2011, 01:37 PM
Replies: 12
Views: 4,992
Posted By CC_seqanswers
Your post is always filled with neat information....

Your post is always filled with neat information. :)

I actually thought of denaturing PE libraries before ePCR like with rapid library. Do not quite understand the necessity of using strepvidin...
Forum: Sample Prep / Library Generation 07-28-2011, 12:54 PM
Replies: 12
Views: 4,992
Posted By CC_seqanswers
I was told the same cut off could be used at...

I was told the same cut off could be used at least in this case. But I am not sure either as it takes some work to validate it.

May I ask a couple of more questions regarding Ampure beads?
1....
Forum: Sample Prep / Library Generation 07-28-2011, 09:47 AM
Replies: 12
Views: 4,992
Posted By CC_seqanswers
Thanks! It made lots of sense. Would you...

Thanks! It made lots of sense.

Would you think this might be one reason to use beads for sizing for 454 PE protocol? It seems to me that beads won't help with the issue. However, for this certain...
Forum: Sample Prep / Library Generation 07-28-2011, 08:35 AM
Replies: 12
Views: 4,992
Posted By CC_seqanswers
I attached two Agilent traces. I marked size...

I attached two Agilent traces. I marked size range and hope it makes sense to you.

After gel sizing and DNA elution, dsDNA was run on DNA chip; and then ssDNA was isolated based on 454 PE...
Forum: Sample Prep / Library Generation 07-27-2011, 02:05 PM
Replies: 12
Views: 4,992
Posted By CC_seqanswers
Thank you, Phillip. I just ran a PE library...

Thank you, Phillip.

I just ran a PE library on 4-12% TBE polyacrylmide gel from Invitrogen and cut between 300-700 and then elute DNA out of the gel. The size range looked very nice on DNA chip...
Forum: Sample Prep / Library Generation 07-26-2011, 03:35 PM
Replies: 14
Views: 57,132
Posted By CC_seqanswers
How about running ssDNA on a ds DNA chip? Is it...

How about running ssDNA on a ds DNA chip? Is it gonna work fine?
Forum: Sample Prep / Library Generation 07-26-2011, 03:32 PM
Replies: 12
Views: 4,992
Posted By CC_seqanswers
Alternative sizing method for 454 PE libraries?

We have had lots of trouble with getting right size of 454 PE libraries. I wonder if one could use gel sizing instead of Ampure beads? Beads sizing always give us inappropriate size or undesired...
Forum: Bioinformatics 03-07-2011, 07:48 AM
Replies: 3
Views: 2,828
Posted By CC_seqanswers
I suspected DNA contamination too and thank you...

I suspected DNA contamination too and thank you for confirming the case!
Forum: Bioinformatics 03-06-2011, 03:14 PM
Replies: 3
Views: 2,828
Posted By CC_seqanswers
high percetage of intergenic reads from RNA-SEQ

We just finished analysis on a RNA-SEQ project, however, we found the exonic reads accounts for only 15% of total reads while intergentic reads almost reached 70%. Very confused. Any thoughts here?
Forum: Bioinformatics 01-24-2011, 07:57 AM
Replies: 12
Views: 7,326
Posted By CC_seqanswers
Sorry, another question. Is Velvet good only for...

Sorry, another question. Is Velvet good only for small genome? The one we are working on is estimated to be around 800M.

Thanks so much!
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