[LOH]

I used this protocol and got this after adapter-PCR:





Can anyone comment? Is it completely crap or not?

To me sample 2 looks better than sample 1, but I have no real clue as in how it should look.

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.

[Runuply]

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.

Hi LOH
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

[mantis]

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?

Hi puraskar theaward,

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.

[paramita7]

How did you dissociate cells from frozen tissue?

Hi guys, I also used that protocol for ATAC-seq from frozen tissue, PCR amplification 5 cycles; and 7 cycles for qPCR(1 to 3rd of Max Fluorescent). It is a little hard for me to control the exact cell number, but after transposition and purification I will use 1ul to check by Qubit, which usually shows around 10ng/ul in 10ul (this should be equal to 25k cells or less). And after I did PCR amplification (in total 5+7 cycles), the purified DNA will be about 50ng/ul in 20ul.
The Bio analyzer is attached.

Hi Runuply,
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,

[Runuply]

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 sequencing I get many background peaks which are very noisy and the peaks at the TSS were not distinct. Anyone has any idea to improve the method and reduce the background peaks?

Hi Wonghe did you solve the problem？
What kind of frozen tissue do you use？ I feel like the cell isolation process is most important.

[Runuply]

Hi Runuply,
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,

Hi paramita7

I am using frozen cartilage for ATAC-seq. I use collagenase to dissociate the chondrocytes.
What kind of frozen tissue do you use?

[paramita7]

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.

[maddy2016]

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.

[seed222222]

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.

Hi maddy,
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.

[milevskiy]

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?

[maddy2016]

Hi maddy,
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.

Yes, I made the ATAC-seq libraries and sent my samples for sequencing. I started with 54,000 cells and performed the TD reaction.
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!

[Vgamma4]

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??

[ScottC]

Does anyone here use NexteraXT rather than Nextera for ATAC-seq library construction?

[jamesdinn]

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.

Hi Runuply & Mantis,
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

[atacseq01]

ATAC-seq: Sequencing prep for Illumina hiseq2000

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.