TITLE: Multi-Seq: my notes from the lab v2
AUTHORS: Luciano Martelotto

[DESCRIPTION]
<div class = "text-blocks"><table border><tr style = "text-align:center;"><td>  </td><td>A</td></tr><tr><td style = "text-align:center;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;">Please note these are just my notes and by no means are ground truth of how Multi-seq needs to be performed. I WELCOME to comments, suggestions and amendments.</td></tr></table></div>

[STEPS]
?. First thing to do is to get very clean nuclei preps. You may start with less clean nuclei if you like but it is likely that CMO will bind to the debris, and that is not good (just a thought). So, if you work with cell lines, iPSCs, PBMCs or some of the ‘liquid’ cancers then nuclei will be pretty clean, but depending on the solid tissues, as you probably have experienced already, you may end up getting dirty nuclei preps.
?. To get clean nuclei you can use either a sucrose gradient or FANS based on DNA content staining DAPI/7-AAD/DRAQ-7(*). I prefer FANS (but this is just me and because I have the sorter in the lab), and for most snRNA-Seq experiments I use DAPI or 7-AAD or DRAQ-7 (I used DAPI more often). After FANS nuclei usually looks super clean and start from there. Sucrose gradient works also quite well, specially to remove myelin from brain tissue, but haven’t used it regularly enough. In any case, starting with clean nuclei (when possible) is good practice to try to have the best and reproducible labelling as possible. (*) These are also dyes are known to not distort chromatin (or very little, as opposed to PI which it does) so we keep this door open for when we do snRNA and snATAC from same sample.Note: FANS based on DNA content is not just an expensive way to clean up nuclei, it can help you identify potential sub-polulation with different ploidy, in particular for cancer samples.
?. It is important that you visually inspect the nuclei preps under the optical microscope (brightfield) before and after sorting. Check for signs of nuclei damage or partially lysed cells. Nuclei sizes and shape vary a lot, so I don’t take this too much into account. I do look at the edges of the nuclei - usually intact nuclei have clear edges. Damaged nuclei are easy to identify with 40x-60x magnification. Sometimes disorganised chromatin popping our out of the nucleus is visible, which is bad sign. Nuclei blebbing is also bad sign.
?. Mix Anchor and Barcoded oligos in 1:1 molar ratio in PBS (no Ca2+/Mg2+) at (*), that is 10 times solution (of the duplex) and incubate at  for . We have been preparing this mix fresh. (*) The working concentration is optional.
What (I think) it's key is to maintain the ratio 40 umol of CMOs (Anchor-Barcoded Oligo and Co-Anchor) per 500,000 cells ( final in ). So for 5x less nuclei, namely 100,000 nuclei, we useCMO, which is 5x less.
0 Room temperature
200 µl
8
?. The number of nuclei you use of of your choice, for example ~100,000 nuclei(*) in Washing Buffer [PBS (no Ca2+/Mg2+) +  BSA (MACS BSA Stock Solution from Miltenyi, Cat. 130-091-376)]. Note: remember that for cells you use BSA or no BSA in your Washing Buffer. If FANS is not an option, then directly resuspend nuclei in 180 uL Washing Buffer and continue with Step 7 below.We usually chose FANS for our nuclei preps clean up step prior to snRNA-Seq (see Frankenstein protocol for snRNAseq using fresh and frozen samples on 10x Platform). (*)Our sorter offset is ~40%, so we sort ~140,000 events to get ~100,000 nuclei. The sorting is done in  of Washing Buffer in 96-well plate (culture plate, round bottom, but LoBind eppis work too). I like culture plates because I always inspect nuclei under the microscope after sorting, so these are ideal.
50 µl
1.5 ml
?. After sorting, transfer the nuclei to a   LoBind eppis tube and complete with chilled Wash Buffer to ~  (Note: when limited nuclei number, I usually use the additional volume to ‘wash’ the well where the nuclei come from and then transfer this wash the respective nuclei in the eppi).
1.5 ml
180 µl
1.5 ml
?. Add  Anchor-Barcode oligo (  =  of ) and gently but thoroughly mix by pipetting using wide-bore tip. So far we’ve maintained the  CMO/500,000 nuclei (CMO/100,000, that's 5x less). According to the authors, for 500,000 nuclei they add of CMO for a final concentration of in final volume). Here I am accounting for 5x less cells, so I add  of CMO in a final volume of ~ (Note: at this stage the Anchor-Barcoded oligo concentration is , but it'll  become when I add the Co-Anchor below (bringing the final volume from  to ). In any case, for 100,000 nuclei Anchor-Barcoded Oligo and Co-Anchor will at the same molarity in the end, that is  or each in final volume, which is 5x less of what's used for 500,000 nuclei, namely ).
8
8
10 µl
40
8
20 µl
200 µl
10 µl
190 µl
190 µl
200 µl
8
200 µl
?. Incubate for at .
4 °C
?. Add Co-Anchor and gently but thoroughly mix.
8
?. Incubate for at .
4 °C
?. Add  of chilled Wash Buffer and mix by inverting 3x.
1 ml
?. Centrifuge: 500 34, 5 min, 4 10
?. Remove supernatant without disturbing the pellet. For this, I always leave behind ~- of supernatant. This is key to avoid losing the nuclei.
20 µl
30 µl
?. Repeat wash for 2 more times using of chilled Wash Buffer. Do not resuspend nuclei after every wash – this help reducing nuclei loss.
1.2 ml
?. In the final wash resuspend in  of chilled Wash Buffer supplemented with -of RNAse Inhibitor (Protector RNAse Inhibitor, Cat. RNAINH-RO, Merck/Roche). One may use RNAse inhibitor all the way, but it becomes expensive. I use RNAse Inhibitor during cell lysis, and in this final resuspension and RNA/cDNA quality looks good.
80 µl
?. If possible, count using Countess II Automated Cell Counter using Trypan Blue (tend to precipitate so it’s a pain. I spin top speed every time I use) or Ethidium Homodimer. Alternatively, proceed with pooling.
?. Combine nuclei from multiple samples to multiplex and filter with a 40-um Flowmi® Cell Strainer.
?. Count using Countess II Automated Cell Counter.
?. Adjust nuclei concentration if needed.
?. Proceed with 10x 3’ v3 protocol.