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Forum: Sample Prep / Library Generation 05-10-2012, 08:49 AM
Replies: 10
Views: 3,644
Posted By RNAseqer
You can't efficiently remove the longer style...

You can't efficiently remove the longer style adapters with columns. That's why beads are required.
Forum: Sample Prep / Library Generation 05-08-2012, 07:44 PM
Replies: 22
Views: 11,847
Posted By RNAseqer
Interesting comments about rRNA removal with...

Interesting comments about rRNA removal with ethanol precipitation. Any experience with removing rRNA while trying to recover only small RNA ?
Forum: Sample Prep / Library Generation 05-08-2012, 07:41 PM
Replies: 8
Views: 5,653
Posted By RNAseqer
Our attempts with TruSeq and diluting adapters...

Our attempts with TruSeq and diluting adapters has so far been unsuccessful. But we have used the Bioo Chip-Seq kit with success. Their Chip adapters are more dilute than their regular DNA adapters.
Forum: Sample Prep / Library Generation 05-08-2012, 07:37 PM
Replies: 22
Views: 9,305
Posted By RNAseqer
We are using Bioo's ChIP-Seq kit with beads. I...

We are using Bioo's ChIP-Seq kit with beads. I believe you have to use beads because the adapters are long and cannot be removed by columns.
Forum: Illumina/Solexa 11-15-2011, 09:12 PM
Replies: 11
Views: 3,626
Posted By RNAseqer
Hi Odile, We use those prep kits too. Many...

Hi Odile,

We use those prep kits too. Many of them are coming near 1 year and they are working very well for us. I bet the short time period is unwarranted.
Forum: Sample Prep / Library Generation 09-27-2011, 05:31 AM
Replies: 3
Views: 3,500
Posted By RNAseqer
According to what I've read anything greater than...

According to what I've read anything greater than a 1x ratio will recover everything <~100. Its only when you go less than 1x do you start being able to size select, ie. .5X or .75X. I'd be...
Forum: Sample Prep / Library Generation 09-27-2011, 05:26 AM
Replies: 22
Views: 9,305
Posted By RNAseqer
We are achieving much better read and quality...

We are achieving much better read and quality scores with Nextflex compared to Truseq. In my experience the libraries are more consistent as well.

We use the Nextflex barcodes for genomic and...
Forum: Sample Prep / Library Generation 05-17-2011, 06:44 PM
Replies: 13
Views: 5,908
Posted By RNAseqer
We're using their indexed adapters for ChIP-Seq,...

We're using their indexed adapters for ChIP-Seq, mRNA-Seq and Genomic sequencing. Heard from our sales rep that they are launching a 48 plex ChIP-Seq kit this month
Forum: Sample Prep / Library Generation 05-17-2011, 06:36 PM
Replies: 6
Views: 4,931
Posted By RNAseqer
how wide of a size range are you able to get away...

how wide of a size range are you able to get away with before sequencing? 300-400 (100 bp) or 300-500 (200 bp) or even more? how does the larger size cause problems with pe reads?
Forum: Sample Prep / Library Generation 03-09-2011, 10:29 PM
Replies: 6
Views: 4,931
Posted By RNAseqer
I know some people are moving away size selection...

I know some people are moving away size selection if they have a tight band when fragmenting their material. Anyone experience this?
Forum: Sample Prep / Library Generation 03-09-2011, 10:21 PM
Replies: 2
Views: 1,467
Posted By RNAseqer
It may be that the salt concentration in your...

It may be that the salt concentration in your initial RNA sample is too high and inhibitory to the RT enzyme. I would purify the RNA with a column and then proceed to RT.
Forum: Sample Prep / Library Generation 03-09-2011, 10:10 PM
Replies: 18
Views: 26,877
Posted By RNAseqer
We're now going to get all our adapters and...

We're now going to get all our adapters and primers from Bioo...
Forum: Sample Prep / Library Generation 03-09-2011, 08:56 PM
Replies: 2
Views: 3,381
Posted By RNAseqer
Size selection after Covaris shearing...necessary?

With a show of hands, how many people still do size selection post ligation after shearing genomic DNA by Covaris ? I've heard that many skip size selection now, but many others still size select.
Forum: Sample Prep / Library Generation 03-09-2011, 08:51 PM
Replies: 1
Views: 4,279
Posted By RNAseqer
ahhh...we used to do this but not anymore. Just...

ahhh...we used to do this but not anymore. Just couldn't get good annealing, we'd see multiple peaks. I recommend using the TruSeq adapters from Illumina or the DNA Adapters...
Forum: Sample Prep / Library Generation 03-09-2011, 08:47 PM
Replies: 5
Views: 7,186
Posted By RNAseqer
i wouldn't do the run with non-annealed adapters....

i wouldn't do the run with non-annealed adapters. it simply won't work. If you're not sure whether you annealed them or not do a qPCR with good positive and negative controls. you'll get your answer...
Forum: Sample Prep / Library Generation 03-09-2011, 08:45 PM
Replies: 28
Views: 13,777
Posted By RNAseqer
anyone find a solution to replace those $5 tubes...

anyone find a solution to replace those $5 tubes by Covaris ?
Forum: Sample Prep / Library Generation 03-09-2011, 08:41 PM
Replies: 11
Views: 9,780
Posted By RNAseqer
barcoded adapters

Looks like Bioo has 48 DNA sequencing adapters (http://www.biooscientific.com/DetailProduct.aspx?itemId=380&CName=Life%20Science&SubName=Next-Generation%20Sequencing&Name=DNA%20Sequencing) with...
Forum: RNA Sequencing 12-22-2010, 10:44 AM
Replies: 39
Views: 16,294
Posted By RNAseqer
Sorry meant to say that the scriptseq kit for...

Sorry meant to say that the scriptseq kit for mRNA has failed our rigorous testing.
Forum: RNA Sequencing 12-22-2010, 10:41 AM
Replies: 39
Views: 16,294
Posted By RNAseqer
We do several 100 mRNA libraries ourselves and...

We do several 100 mRNA libraries ourselves and have been looking into kit alternatives for some time. The scriptminer kit from epicenter has failed in our hands. We couldn't get libraries. Right now...
Forum: Sample Prep / Library Generation 10-20-2010, 11:13 PM
Replies: 1
Views: 1,980
Posted By RNAseqer
mRNA library problems

I'm having a lot of issues with NuGEN's Ovation 3'DGE and RNA-Seq library preparation kits. No matter what I do, I can't seem to generate any libraries for sequencing. Has anyone had a similar...
Forum: SOLiD 09-16-2010, 03:12 PM
Replies: 18
Views: 10,548
Posted By RNAseqer
I've heard that trimming is not as efficient as...

I've heard that trimming is not as efficient as alignment in the pipeline and then building up or down from there.
Forum: Illumina/Solexa 09-16-2010, 03:06 PM
Replies: 7
Views: 23,397
Posted By RNAseqer
mirna barcoding

We just performed a huge investigation into the barcoding of small RNAs on the 3'adapter and these are our results:
By intelligently designing the barcode sequence so that there is even spacing...
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