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Thread | Thread Starter | Forum | Replies | Last Post |
ATAC seq lysis time | zoe561 | Sample Prep / Library Generation | 3 | 03-05-2015 09:00 AM |
Error in RNA-seq analysis of Arabidopsis following how-to- seq wiki protocol | himanshu04 | RNA Sequencing | 1 | 03-23-2012 06:58 AM |
Anyone has protocol for Ion PES protocol? | marcowanger | Ion Torrent | 1 | 01-18-2012 08:28 PM |
illumina alternative v1.5 protocol for small rna seq vs. the standard protocol | ik76 | Sample Prep / Library Generation | 1 | 03-25-2010 02:24 PM |
mRNA-Seq protocol | Xi Wang | Illumina/Solexa | 5 | 11-05-2009 06:41 AM |
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#41 |
Junior Member
Location: living in the lab in Europe Join Date: Jan 2016
Posts: 5
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Hi Runuply,
Yes, I did a 1.6V ampure treatment: The small primers won't bind to the peaks with this DNA/beads ratio. Good luck! |
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#42 | |
Junior Member
Location: Japan Join Date: Feb 2016
Posts: 6
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Thanks Mantis. I did the AMpure , it looks good. By the way, may I know that, normally how much DNA can you get after lysis if you use 50K cells? Do you check the concentration before PCR amplification. |
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#43 |
Junior Member
Location: living in the lab in Europe Join Date: Jan 2016
Posts: 5
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#44 | |
Junior Member
Location: San Antonio Join Date: Feb 2016
Posts: 2
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So I tried using a AmpureXP x1.8V cleanup. It cleared the band. |
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#45 | |
Junior Member
Location: San Antonio Join Date: Feb 2016
Posts: 2
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Hi Mantis,
I got similar BA image post AmpureXP cleanup. ![]() ![]() 1] I was wondering what was the conversion factor you used to calculate the DNA concentration of the libraries for the cluster generation. ![]() 1.1 Did you use the broad range like 150-1200bp in BA and found out the pM concentration from BA region table? or 1.2 used some conversion tables for the ng/uL for each region of library? or 1.3 Did a Qubit HS DNA concentration of the libraries? 2] Illumina tells to normalize libraries to 2nM before adding them to cBOT prep tubes. Did you normalize to 2nM? If so did you use Tris-HCl pH8.5 with 0.1% Tween 20 or used the regular TE buffer which had DNA eluted post Ampure XP purification. 3] Did you use phiX control at 1-5% before loading on the cBot? Quote:
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#46 |
Registered Vendor
Location: CA Join Date: Jul 2010
Posts: 23
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You have huge peaks at size > 1kb region, indicating your libraries are over enriched with fragments >1kb. Fragments >1kb will decrease the sequencing efficiency and is not suitable for Illumina sequencer.
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#47 | |
Junior Member
Location: Japan Join Date: Feb 2016
Posts: 6
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Could you please tell us, what should be the factors to get large size libraries sizes over 1kb. Do you think it is under-tagmentation, cell number dependent? Best Runuply |
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#48 | |
Junior Member
Location: living in the lab in Europe Join Date: Jan 2016
Posts: 5
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To answer your questions: 1.1 Yes, I used the full range. So, my average size was around 450-500 for each sample; I calculated the molarity from that to obtain eventually 4 nM in the end. 1.2 So, I did not specify for every region of the library. 1.3 I indeed measured the concentration with the Qubit. For questions 2 en 3 I unfortunately cannot help you. I handed in pooled 4 nM libraries to our sequencing facility. (I elute my DNA from the beads with MilliQ.) The subsequent dilutions and processing are done by the facility. |
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#49 | |
Junior Member
Location: atlanta Join Date: Jan 2016
Posts: 6
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i am also doing ATAC-seq from froze tissue. Can you give me an idea how you dissociate cells from tissue? I am having difficulty with it. Thank you, |
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#50 | |
Junior Member
Location: Japan Join Date: Feb 2016
Posts: 6
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What kind of frozen tissue do you use? I feel like the cell isolation process is most important. |
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#51 | |
Junior Member
Location: Japan Join Date: Feb 2016
Posts: 6
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I am using frozen cartilage for ATAC-seq. I use collagenase to dissociate the chondrocytes. What kind of frozen tissue do you use? |
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#52 |
Junior Member
Location: atlanta Join Date: Jan 2016
Posts: 6
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Hi Runuply,
I have frozen brain tissue and I am just using grind and strain method. But after purification steps I am loosing a lot of DNA. |
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#53 |
Junior Member
Location: Columbus, Ohio, USA Join Date: Jun 2016
Posts: 3
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Hi All, I am also planning to start ATAC-seq using fresh cells. My question is how do I harvest the cells..is it by scraping cells in 1x cold PBS and then counting them on hemocytometer to make sure how many cells I am staring with. Also what should be the best internal control during sequencing? I assume cells without treatment with Transposase? Any help is highly appreciated. Thanks in advance.
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#54 | |
Junior Member
Location: 7500 Brompton road Join Date: Oct 2016
Posts: 1
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I am also trying to perform ATAC-seq on fresh cell. Did you solve the cell collection problem? Are you use trypsin or harvest cell by scraping? Thank you. |
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#55 |
Junior Member
Location: Australia Join Date: Oct 2016
Posts: 1
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I have tried the standard protocol on COMMA-D mouse mammary cell line with limited success. We appear to have a log of smaller fragments and not getting the nice banding pattern typical of this technique. I have tried to optimise the TD reaction but that seems to have little effect. Has anyone gotten this to work on cell lines or cells which might be harder to lyse than blood cells?
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#56 | |
Junior Member
Location: Columbus, Ohio, USA Join Date: Jun 2016
Posts: 3
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For collecting cells, first I discarded the media, washed cells in PBS twice. Added TryPLE to detach cells and scraped them gently in regular media. Followed by counting cells in hemocytometer. Hope this helps. Good luck! |
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#57 |
Junior Member
Location: Europe Join Date: May 2016
Posts: 2
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Hi ATAC-fanatics.
Since my experience with ATAC is only 2 weeks old, I have a question regarding the quality of my ATAC library. I used 50.000 cells and followed the instruction as described in Buenrostro et al. The Bioanalyzer gave me the following picture. It seems the DNA size of the libraries is quite small, no? Do you guys think that its sequenceable?? |
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#58 |
Senior Member
Location: Monash University, Melbourne, Australia. Join Date: Jan 2008
Posts: 246
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Does anyone here use NexteraXT rather than Nextera for ATAC-seq library construction?
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#59 | |
Junior Member
Location: Copenhagen Join Date: Dec 2016
Posts: 2
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I'm new user for ATAC-seq experiment, i see that you use AMpure beads to clean DNA, could i use it for the each purification step (after 30 min tagmentation, after PCR cycles...?) or only before loading in Bioanalyser? thank you |
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#60 |
Junior Member
Location: Texas Join Date: Jan 2017
Posts: 1
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Hi all -
For those of you who prepare ATAC libraries followed by sequencing on Illumina hiseq2000 - what specific illumina-based protocols and reagents are you using? (for cbot, Hiseq, etc.)? thanks. |
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