Thread by @LGMartelotto

Dear #singlecell global team (#scGT), it was a very inspiring week for #scQA. @hoheyn and I would like to THANK to the @cmcginnisUCSF for his comprehensive Tweetorial on #MultiSeq.

For the Feb3/2020 #scQA/#scQC Forum (w/@hoheyn &
@LGMartelotto) we'll discuss some #snATAC-Seq!

First a bit of history...

Assay for Transposase-Accessible Chromatin (ATAC-Seq) was originally published in in 2013 (@naturemethods PMID: 24097267) by @JD_Buenrostro in the labs of H. Chang and W. Greenleaf at @Stanford.

ATAC-Seq is a VERY efficient technique, and was quickly implemented at #singlecell level in 2015 by the same team (@nature PMID: 26083756). These are good reads and help understand why this method is so revolutionary.

At the heart of any version of #ATAC-Seq is the MAGICAL #Tn5 that does exactly what we want it to do: it goes inside the nucleus, finds regions of open chromatin, performs efficient transposition and introduces sequencing adapters. In addition, #ATAC-Seq is VERY versatile.

For this week's #scQA we will focus (mainly) on sample prep for #ATAC-Seq applied to sc or sn. Around mid week, I'll share some tips/tricks to do snRNA/ATAC from same nuclei preparation for minute samples for @10xGenomics platform. I'll also comment on FANS for snATAC-Seq.

As the week moves along, of course feel free to comment on other aspects involving ATAC-Seq or versions of it (e.g joint expression/ATAC profiling like SNARE-Seq, sci-CAR or scNMT-seq; or alternatives like Cut&Tag or ACT-Seq). Share papers/experiences with us.

In terms of sample preparation for sc/snATAC-Seq, there are many protocols in protocols.io and within published papers for sample processing for good nuclei prep, which is key aspect for successful ATAC procedure irrespective of platform (#Chromium, #OmniATAC, etc).

Three of these protocol ATAC protocol lysis buffer (0.1% NP40)12, the #OmniATAC ~ @10xGenomics protocol lysis buffer (0.1% NP40, 0.1% Tween-20 and 0.01% Digitonin), and the #Takaku-ATAC protocol (0.1% Triton X-100).

In PMID: 30679562 the authors improved Omni-ATAC by including of 0.1%Tween-20 and 0.01% Digitonin during the transposase reaction step (i.e. OPTI-ATAC) and used this method to successfully process and perform #snATAC-Seq cryopreserved tissues samples.

Late in 2019, Andrew Adey lab (@acadey80), shared a pre-print (biorxiv.org/content/10.110…) where they used a small molecule inhibitor Pitstop 2™ to increase the ability of transposase to enter the nucleus and generate highly complex single-cell libraries (e.i. scip-ATAC-seq).

In my lab, the #Chromium Single Cell ATAC has been the battle horse of many projects. It works well, it's robust, very easy to use, but $$ and not easy to 'hack' (😜). @10xGenomics offers some guidance and general tips/tricks for nuclei prep for PBMCs, cell lines and mouse brain.

We have been using an in-house protocol to prepare nuclei from small snap/flash frozen, cryopreserved or fresh solid tissues biopsies to run snRNA-Seq and ATAC-Seq from the nuclear prep. This is great when sample is limiting and you want your data to match nicely.

I will share some tips/trick, DOs and DONT's around mid week. In brief, we use #FANS and have been used DAPI with minimal or non alteration in the data. PI is a no go!!! (others no go are: Ethidium Homodimer, Vybrant Dyecycle Green, Zombie Violet).

I'm running a side-2-side snATAC-Seq comparison of DAPI/DRAQ-7/7-AAD/PI/None. I'm also considering SYTO™ RNASelect™ Green Fluorescent, which stains RNA.
Will share when this is done.

Please share your thoughts, opinion about sample processing and preparation methods for sc or snATAC-Seq. More information will come soon in this thread. If you have used snATAC-Seq before or you want to start, this is the place to share, ask and troubleshoot.

*to the amazing @cmcginnisUCSF ;-)

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