[Alpaca]

Hi,
Did you end-up sequencing either of these libraries shown in the bioanalyser traces? Did you get good results?
We have libraries with a relatively large peak at around 55bp (we think its primer, and it's as big as the 200bp peak) and wondered if the presence of this would ruin a sequencing run. Does anyone have any insights to offer, please? We have low sample amount so need to avoid another clean-up step if we can, but don't want to sequence rubbish either :-).

Thanks!

Kylie

[Zaag]

Hi,
Did you end-up sequencing either of these libraries shown in the bioanalyser traces? Did you get good results?
We have libraries with a relatively large peak at around 55bp (we think its primer, and it's as big as the 200bp peak) and wondered if the presence of this would ruin a sequencing run. Does anyone have any insights to offer, please? We have low sample amount so need to avoid another clean-up step if we can, but don't want to sequence rubbish either :-).

Thanks!

Kylie

I have sent some of them out this morning, I don't know when I get my results, but I will post the bio-analyser traces and some sequencing QC when I get it back.

[Alpaca]

Thanks for speedy the update, and good luck with the sequencing!

Has anyone else seen the same thing (50-70bp peak) and got good sequencing results (i.e. real sequence not sequenced-artifacts)?

Kylie

[finixtree]

Don't put too much effort to get a good bioanalyzer trace.
Bioanalyzer will give funny trace, try TBE page gel.
I know, in theory, bioanalyzer should work well, but in fact not.

Hi Wonghe,

I am very interested to see bioanalyzer trace of a successful ATAC-Seq experiment. If possible, would you please share your bioanalyzer trace. If not, would you please describe how was the pattern.

[Celticray]

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.

Hi Zaag,
were you be able to get a good sequencing run/result on you sample 2? It's my first time doing the ATAC-Seq and i have no clue how the Bioanalyzer result should look like.

[mahoney]

We have been having mixed results running these samples on HS-DNA kit. Some work others seem to clog the chip. Does anyone have any suggestions on standard protocol for running these with regards to concentration. I've skipped wells....sometimes it works other times it makes no difference.

[mantis]

Gel of good ATAC-seq experiment

hi everyone,

I just tried my first ATAC-seq on cells and using the protocol of Buenrostro and I obtain good results: sharp peaks, low background, (appr. 50000 cells).

I included my HS picture.

After measuring the molarity for each fragment, I obtained a good nucleosome distribution (also indicated on the gel, in the right). Before putting on the HS chip, I Ampured (1.6V) the sample.

[Runuply]

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.

[mantis]

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 10l (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.

Looks good Runuply: there seems to be a good nucleosome pattern on the gel picture. I think I would advice you to try to get rid of the primer peak at 65 before submitting the sample for sequencing.

[Runuply]

Hi mantis, thanks for your reply. This is my 2nd test.
May I know that how do you get rid of the primer peak? Do you use something like Ampure purification method?

[mantis]

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!

[Runuply]

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!

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.

[mantis]

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.

I never did...

[puraskar_theaward]

I have sent some of them out this morning, I don't know when I get my results, but I will post the bio-analyser traces and some sequencing QC when I get it back.

I also got the similar peak at the 55bp. Ppl reported that to be either the adapter 50-53bp.

So I tried using a AmpureXP x1.8V cleanup. It cleared the band.

[puraskar_theaward]

What should be the concentration of the DNA library for Clustering

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 everyone,

I just tried my first ATAC-seq on cells and using the protocol of Buenrostro and I obtain good results: sharp peaks, low background, (appr. 50000 cells).

I included my HS picture.

After measuring the molarity for each fragment, I obtained a good nucleosome distribution (also indicated on the gel, in the right). Before putting on the HS chip, I Ampured (1.6V) the sample.