TITLE: Multi-Seq: my notes from the lab
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. [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[or ] of CMOs (Anchor-Barcoded Oligo and Co-Anchor) per 500,000 cells ( [or ] ) final in ). So for 5x less nuclei, namely 100,000 nuclei, we use[or ] CMO, which is 5x less.
0 Room temperature
0 Room temperature
40
100
200 µl
8
20
?. 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). For 500,000 nuclei 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 ).[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). For 500,000 nuclei 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
20
20
10 µl
100
20
20 µl
200 µl
10 µl
190 µl
190 µl
200 µl
20
200 µl
?. Incubate for at .
4 °C
?. Add  [or] Co-Anchor and gently but thoroughly mix.
8
20
?. 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.
?. After GEM formation and RT proceed as per 3' v3.x workflow until cDNA amplification. Elute cDNA in .
35 µl
?. Transfer  of cDNA onto cDNA Amplification mix supplemented with  of  of Multi-Seq Additive primer.
34 µl
1 µl
?. Perform cDNA amplification and 0.6X SPRI clean-up according to  3' v3.x workflow.
?. Endogenous transcript cDNA is bound to the SPRI beads. Continue protocol for endogenous transcripts without change.
?. Add 1.4X SPRI to supernatant to obtain a final SPRI volume of 2X SPRI. For example, the cDNA PCR reaction volume is , therefore add another  of SPRI to the supernatant which already contains 60 μl of SPRI. The final volume will be  containing 2X SPRI. Transfer entire volume into a low-bind 1.5 mL tube and Incubate for at room temperature.
100 µl
140 µl
300 µl
?. Place tube on magnet and wait ~ until solution is clear and then carefully remove and discard the supernatant.
?. Add 400 μl 80% Ethanol to the tube without disturbing the pellet and stand for (only one 80% Ethanol wash). Carefully remove and discard the ethanol wash.
?. Centrifuge tube briefly and return it to magnet. Remove and discard any remaining ethanol.
?. Resuspend in beads in  water.
50 µl
?. Perform another round of 2X SPRI purification by adding  SPRI reagent directly onto resuspended beads. Mix by pipetting and incubate  at room temperature.
100 µl
?. Place tube on magnet and wait ~until solution is clear. Carefully remove and discard the supernatant.
?. Add 80% Ethanol to the tube without disturbing the pellet and stand for  (1st Ethanol wash). Carefully remove and discard the ethanol wash.
200 µl
?. Add  80% Ethanol to the tube without disturbing the pellet and stand for (2nd Ethanol wash). Carefully remove and discard the ethanol wash.
200 µl
?. Centrifuge tube briefly and return it to magnet. Remove and discard any remaining ethanol and allow the beads to air dry for . Do not over dry beads.
?. Resuspend beads in  water or EB. Pipette mix vigorously and incubate at room temperature for .
40 µl
?. Place tube on magnet and transfer clear supernatant into one PCR tube.
Quantification of barcode DNA concentration using Qubit (typical range is 0.5 - 5 ng/µL) is recommended but it's optional. -If quantifying the use   of material for indexing PCR. -Alternatiovely use   of material for indexing. -The use of the whole material is also possible but some primer optimization may be needed as you PCR bubbles due to over-cycling is likely (i.e. primers get exhausted). When this happen, run a P5/P7 reaction for one-2 cycles.
?. Set up the PCR reaction as follows: Kapa HiFi HotStart ReadyMix (2X) 10 µM SI Primer (e.g. 3' v3.1, PN 2000095) 10 µM RPIx primer (choose unique RPI for each sample from 10X lane) barcode DNA or  or entire volume (~)Nuclease-free water tofinal volume.Cycling conditions:1.  x 2.  x 3.  x 4.  x Go to 2 for ~6-10 cycles5.  x 6. Hold at .
50 µl
5 µl
5 µl
3.5 ng
5 µl
40 µl
50 µl
95 °C
98 °C
60 °C
72 °C
72 °C
4 °C
?. Add  (1.6X) SPRI to each PCR product, pipette mix thoroughly. Incubate at room temperature for .
160 µl
?. Place tube on magnet (High), wait for solution to clear. Remove and discard supernatant.
?. Wash beads twice on magnet (High) with  of 80% ethanol and allow to stand for  between washes.
200 µl
?. After second wash, briefly centrifuge beads and place on magnetic rack (Low). Remove remaining ethanol with P20 micropipette. Air-dry beads on magnet for . Do not over dry beads.
?. Remove from magnet, resuspend beads in  buffer EB and pipette mix thoroughly to resuspend. Incubate at room temperature for .
25 µl
?. Return to magnet (Low), wait for solution to clear, and transfer supernatant to PCR strip tube.
?. Quantify barcode library concentration (1:5 - 1:10) using Bioanalyzer High Sensitivity DNA analysis. See representative electropherogram below.
?. Barcodes can be sequenced independently or as fraction of endogenous cDNA library. Aim for ~3000-5000 read pairs per cell.
?. For analysis go to https://github.com/chris-mcginnis-ucsf/MULTI-seq.