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Forum: Sample Prep / Library Generation 08-26-2015, 05:43 PM
Replies: 69
Views: 44,391
Posted By Wonghe
Yes I get my atac to work on 70K freshly...

Yes I get my atac to work on 70K freshly trypsinize cells using the original Greenleaf protocol. Depending on the cell type you are working on you might need to adjust the lysis buffer content to...
Forum: Sample Prep / Library Generation 08-20-2015, 07:34 PM
Replies: 12
Views: 5,105
Posted By Wonghe
low mapping rate

Hi, I constructed a RNA-seq library using the clontech Smarter total stranded RNA low input kit. However after mapping using BWA and trimming the adapters I only manage to get 50% mapping rate.
I'm...
Forum: Sample Prep / Library Generation 05-19-2015, 01:37 AM
Replies: 69
Views: 44,391
Posted By Wonghe
frozen tissue

Hi All, I've been trying to do ATAC-seq on nuclei isolated from frozen tissues with sucrose gradient. Although I am able to obtain a nucleosome-like pattern (see above attachment) but after...
Forum: Sample Prep / Library Generation 12-30-2014, 06:37 PM
Replies: 69
Views: 44,391
Posted By Wonghe
A correction to my previous post, tagmentation...

A correction to my previous post, tagmentation was done at 37C for 30min in the below conditions:
1) 20K cells, 10ul Tag reaction vol (1ul enzyme) no clean up before PCR (10cycles) clean up using...
Forum: Sample Prep / Library Generation 12-29-2014, 11:05 PM
Replies: 69
Views: 44,391
Posted By Wonghe
I have been trying the ATAC-seq protocol but fail...

I have been trying the ATAC-seq protocol but fail to get the nucleosome pattern. Can anyone help?

I collected my fresh cells from the culture plate by scraping them off in PBS where I split them...
Forum: Sample Prep / Library Generation 11-24-2014, 12:19 AM
Replies: 6
Views: 3,930
Posted By Wonghe
I also got a smear for my PCR product between...

I also got a smear for my PCR product between 150-2500bp. Has anybody sequenced these DNA does it enriched the open chomatin?
Forum: Bioinformatics 11-18-2014, 01:23 AM
Replies: 649
Views: 150,969
Posted By Wonghe
Thank you for the suggestion. We have trimmed...

Thank you for the suggestion. We have trimmed both the 5' and 3' ends to remove base with poor quality. Hope this will help increase the overall mapping rate.
Forum: Bioinformatics 11-17-2014, 10:13 PM
Replies: 649
Views: 150,969
Posted By Wonghe
low mapping rate in SR reads

Hi,
We recently encounter very poor mapping efficiency for our RRBS libraries sequenced with Hi-Seq Rapid mode 1x51bp. Using the default parameter in Bismark we only get 9-45% mapping efficiency. We...
Forum: Sample Prep / Library Generation 10-01-2014, 07:34 PM
Replies: 69
Views: 44,391
Posted By Wonghe
Hi, I just downloaded the protocol and is...

Hi,
I just downloaded the protocol and is wondering if anyone here has tested it using the Nextera DNA or XT kit? Does it make a difference?
Since there is no size selection before sequencing how...
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