TITLE: snATAC-Seq on 10x ChromiumTM platform for fresh, frozen and cryopreserved material: my notes from the lab (UPDATED VERSION)
AUTHORS: Luciano Martelotto

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>-Here, I will </span><span style = "font-weight:bold;">only</span><span> include tips and notes related to snATAC workflow. Thus, I assume you are familiar with Frankenstein protocol below:</span></div></div><div class = "text-block">- These are ONLY a collection of my lab notes. This is not a protocol.</div><div class = "text-block"><div class = "justify" style = "text-align:left">-These notes are derived from trial and error in order to get successful runs applied to fresh, snap/flash and cryopreserved frozen cell lines as well as to tissue derived from solid tumours and other tissues using Chromium Platform (10x Genomics).</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">-Be mindful these are just my notes and by no means are ground truth of how snATAC-Seq needs to be performed. I WELCOME to comments, suggestions and amendments.</div></div></div>

[STEPS]
?. [snATAC-Seq]
Sort as many nuclei as possible into a round-bottom 96-well plate well containing  of ice-cold ATAC Wash Buffer-Dig.
Ideally, perform the following steps WHILE the RT reaction is RUNNING. See Frankenstein protocol for nuclei prep. The steps below assumes the nuclei input for snATAC is relatively low.
100 µl
DO NOT, I repeat DO NOT sort into Diluted Nuclei Buffer.
Avoid excessive number of steps to reduce nuclei loss. Work as quicky as you can. Always leave some volume (10-15 uL) of supernatant behind when washing.
Note there will be significant nuclei loss during washes and nuclei counting, so you may want to make sure the washing steps are done carefully. Take into account this loss when deciding aimed nuclei. To reduce loss, follow the tips below (bold).
?. [snATAC-Seq]
Transfer the entire volume to 0.2 ml PCR tube (LoBind!).
?. [snATAC-Seq]
Add  of ATAC Wash Buffer-Dig to the well.
50 µl
?. [snATAC-Seq]
Transfer any remanent nuclei to the 0.2 ml PCR tube (~ ).
150 µl
?. [snATAC-Seq]
Centrifuge the nuclei at  for  at .
Centrifuge: 500 34
4 °C
?. [snATAC-Seq]
Remove supernatant leaving behind ~ .
10 µl
?. [snATAC-Seq]
Gently add  ice-cold Diluted Nuclei Buffer.
100 µl
DO NOT resuspend nuclei. This is just a buffer exchange step.
?. [snATAC-Seq]
Centrifuge the nuclei at  for  at .
Centrifuge: 500 34
4 °C
?. [snATAC-Seq]
Remove  of the supernatant in two steps, namely, remove  first and then  (pellet may not be visible!).
100 µl
90 µl
10 µl
?. [snATAC-Seq]
Gently add  ice-cold Diluted Nuclei Buffer.
100 µl
DO NOT resuspend nuclei. This is just a buffer exchange step.
?. [snATAC-Seq]
Centrifuge the nuclei at  for  at .
Centrifuge: 500 34
4 °C
?. [snATAC-Seq]
Remove the supernatant (~ ) in two steps, namely, remove  first and then as much volume as possible to leave leaving behind ~  —  (avoid disturbing pellet).
100 µl
90 µl
7 µl
10 µl
If you manage to remove the whole volume without disturbing the pellet, then add   of Diluted Nuclei Buffer and proceed to step 13.
?. [snATAC-Seq]
Resuspend nuclei in the ~  —  of ice-cold Diluted Nuclei Buffer, carefully washing walls of the tube to ensure all nuclei are in solution.
7 µl
10 µl
?. [snATAC-Seq]
Take  —  and dilute 1:5 with Diluted Nuclei Buffer.
1 µl
2 µl
?. [snATAC-Seq]
Mix 1:1 with Trypan Blue and count the number of nuclei with a cell counter (Countess II FL Automated Cell Counter) or hematocytometer (the counting is to have an idea of how many nuclei to expect based on the recovery factor).
Note that Trypan Blue (TB) tend to precipitate, so centrifuge vial of TP at top speed and use supernatant for staining.
?. [snATAC-Seq]
Inspect under the microscope. Nuclei should look clean and intact. See attached photo:
?. [snATAC-Seq]
Take  of nuclei in Diluted Nuclei Buffer and proceed directly to Chromium Single Cell ATAC Reagent Kits protocol (CG000168 Rev A). The volume added to the Transposition reaction will vary; for low input samples we usually use all  of nuclei prep. Alternatively, follow recommendations of the User Guide to estimate volume of nuclei to add to recover a determined targeted nuclei recovery (Page 20, CG000168 Rev A).To estimate the Number of Recovered Nuclei, do the following calculation: [Nuclei Concentration (from step 35) x Volume of Nuclei (up to 5 μl)] / 1.53 (recovery efficiency factor)
5 µl
5 µl