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Search: Posts Made By: JHU-ChIPmaniac
Forum: Epigenetics 06-23-2011, 09:53 AM
Replies: 4
Views: 2,878
Posted By JHU-ChIPmaniac
Definitely do qPCR on your libraries to make sure...

Definitely do qPCR on your libraries to make sure you still have good enrichment following library construction. I have made libraries from ChiP samples that were good (high fold enrichment of...
Forum: Sample Prep / Library Generation 05-13-2011, 01:11 PM
Replies: 13
Views: 5,901
Posted By JHU-ChIPmaniac
I don't have an answer for you but I'm also...

I don't have an answer for you but I'm also interested in multiplexing ChIP-seq libraries. I was going to try the Tru-seq kit. What kind of problems are you running into?
Forum: Illumina/Solexa 05-09-2011, 06:54 AM
Replies: 7
Views: 4,493
Posted By JHU-ChIPmaniac
I'm not yet using the TruSeq protocol but plan on...

I'm not yet using the TruSeq protocol but plan on it in the future. I'll have to keep all this in mind.
Forum: Core Facilities 05-05-2011, 09:37 AM
Replies: 9
Views: 7,714
Posted By JHU-ChIPmaniac
Any Experience with Otogenetics

We are sending a genomic DNA sample to Otogenetics for human exome sequencing. They will make the library, sequence and do some bioinformatics at a very reasonable intro price. They are also giving...
Forum: Sample Prep / Library Generation 05-05-2011, 09:03 AM
Replies: 2
Views: 2,235
Posted By JHU-ChIPmaniac
I replied on your other thread.

I replied on your other thread.
Forum: Illumina/Solexa 05-05-2011, 09:01 AM
Replies: 7
Views: 4,493
Posted By JHU-ChIPmaniac
Are you using the Egel specific ladders from...

Are you using the Egel specific ladders from Invitrogen? I use the 50 bp Egel ladder for my size selection of ChIP products and the isolated fragments correlate well with what we see on the...
Forum: Illumina/Solexa 04-21-2011, 09:51 AM
Replies: 76
Views: 44,252
Posted By JHU-ChIPmaniac
I don't usually do a PCR cleanup but if I do I...

I don't usually do a PCR cleanup but if I do I just use the Qiagen PCR cleanup but this is usually just to reduce the volume so I can load everything into 1 well.
Forum: Illumina/Solexa 04-20-2011, 12:17 PM
Replies: 76
Views: 44,252
Posted By JHU-ChIPmaniac
I also use the E-gel 2% size select gels...

I also use the E-gel 2% size select gels following amplification. This gets rid of all the adaptor species in the libraries. I typically have plenty of library for sequencing.
Forum: Epigenetics 10-22-2010, 08:42 AM
Replies: 5
Views: 4,183
Posted By JHU-ChIPmaniac
I don't have any experience with small cell...

I don't have any experience with small cell numbers. Are you pulling down a histone modification or a TF? The only publications I have seen using low cell numbers (10,000 cells) and successful...
Forum: Epigenetics 09-01-2010, 12:14 PM
Replies: 13
Views: 7,524
Posted By JHU-ChIPmaniac
Thanks GKM, I did not analyze the data...

Thanks GKM,

I did not analyze the data myself so I will have to ask our informatics person these questions. He is using CisGenome since he is the one who wrote the program. Do you think I should...
Forum: Epigenetics 09-01-2010, 12:04 PM
Replies: 13
Views: 7,524
Posted By JHU-ChIPmaniac
Just wanted to add that this particular Myc...

Just wanted to add that this particular Myc antibody has been used by multiple labs for a number of years. There is a boat load of ChIP-chip and ChIP-seq data out there for Myc using this antibody...
Forum: Epigenetics 09-01-2010, 12:01 PM
Replies: 13
Views: 7,524
Posted By JHU-ChIPmaniac
The libraries are made from ChIP products...

The libraries are made from ChIP products generated from a human cell line. Myc, the TF we study is known to bind to thousands of promoters and maybe 4000-5000 intergenic sites. My chromatin was...
Forum: Epigenetics 09-01-2010, 10:57 AM
Replies: 13
Views: 7,524
Posted By JHU-ChIPmaniac
Hi GKM, After ChIP but before library...

Hi GKM,

After ChIP but before library construction, I do qPCR and calculate a %TI for my TF antibody and for an IgG control. For a number of target promoters I see anywhere from 10 to 100's of...
Forum: Epigenetics 09-01-2010, 09:17 AM
Replies: 13
Views: 7,524
Posted By JHU-ChIPmaniac
I have just encountered the same problem. Been...

I have just encountered the same problem. Been doing ChIP for years with the same antibody for a TF and it usually works beautifully. After making the libary, I validated 3-4 promoters that I know...
Forum: Sample Prep / Library Generation 07-29-2010, 10:26 AM
Replies: 1
Views: 1,811
Posted By JHU-ChIPmaniac
Adaptors for ChIP-seq libraries

I have made a couple of ChIP-seq libraries using Illumina's DNA library kit. I diluted the adaptors 1:30 to compensate for much lower DNA input (10-20ng). I will be getting my sequencing data back...
Forum: Introductions 07-28-2010, 07:33 AM
Replies: 0
Views: 903
Posted By JHU-ChIPmaniac
Hi from Baltimore

I'm now to Seq Answers but have already gotten a ton of info. Thanks for the site. I've been doing ChIP for many years but am just now venturing into the ChIP-seq world. I'm do to get my first...
Forum: Sample Prep / Library Generation 07-28-2010, 07:18 AM
Replies: 6
Views: 5,147
Posted By JHU-ChIPmaniac
There was some discussion about this on another...

There was some discussion about this on another thread. The thought was that it may be due to overamplification of the adaptor ligated fragments. I use the Invitrogen E-Gel size select system to...
Forum: Sample Prep / Library Generation 07-28-2010, 07:10 AM
Replies: 2
Views: 3,802
Posted By JHU-ChIPmaniac
I haven't done ChIP in bacteria but once you have...

I haven't done ChIP in bacteria but once you have the ChIP material I would think making the libraries would be the same. I'm waiting to get back sequencing data from my first ChIP seq libraries. ...
Forum: Sample Prep / Library Generation 07-13-2010, 10:42 AM
Replies: 13
Views: 7,857
Posted By JHU-ChIPmaniac
We actually have a bioruptor that works very well...

We actually have a bioruptor that works very well but this cup horn looks very interesting. We won't be investing anymore in sonication equipment right now but i will pass on this info to other labs...
Forum: Sample Prep / Library Generation 07-13-2010, 10:33 AM
Replies: 0
Views: 1,847
Posted By JHU-ChIPmaniac
New to ChIP-seq

Hi everyone:

I posted this same thing on another forum but thought it most useful here (sorry for the double post)

I've been lurking here for awhile and have finally decided to post. I have...
Forum: Illumina/Solexa 07-13-2010, 10:29 AM
Replies: 0
Views: 1,250
Posted By JHU-ChIPmaniac
New to ChIP-seq

Hi everyone:

I've been lurking here for awhile and have finally decided to post. I have read many of the threads on generation of ChIP-seq libraries and have gained considerable knowledge about...
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