Multiome Tips | CSHL Single Cell Bio

So you think you want to do a 10x Multiome Experiment?

I’m already doing scRNA-seq. What’s the difference?

If you already have working sample prep protocols for scRNA-seq you’re off to a good start – the main reason that Multiome sample prep is different is that it requires nuclei to make the ATAC-seq part of the assay successful.

What do good nuclei look like?

Should be mostly single nuclei (not clumps)

Can see there is little other debris

This sample is diluted 1/5 – this would be a good

multiome concentration (approximately 3,100 nuclei/ul)

How do I add a nuclei isolation step?

Where in your sample prep you add your nuclei isolation step depends on a few key things:

Do you need to enrich for a specific cell type?

Must this enrichment be done on whole cells or is it a nuclear marker?

If you do need to do an enrichment that is dependent on whole cells as input, you have to do your nuclei isolation after. The main challenge this will create is that you will need a much larger number of enriched cells than you do for scRNA-seq because you can expect to lose many of them during the nuclei isolation and concentration steps. Further discussion of the caveats of this issue can be found below in the section: Problem #1.

If you do not need to do an enrichment or you can enrich with a nuclear marker it may be worthwhile to consider a nuclei isolation protocol that starts with whole tissue instead of beginning with a dissociation step. There are many protocols designed to isolate nuclei from tissue including a nuclei isolation kit from 10x which is compatible with snRNA-seq, scATAC-seq and scMultiome experiments. Depending on the composition of the tissue you may face different issues. If the tissue has RNase containing cells, you may face Problem #2 whereas if your tissue produces large amounts of debris during your nuclei isolation you may face Problem #3.

If you’re already doing snRNA-seq you’re off to an even better start. The main problem you may face is Problem #1 the low input problem, since the tagmentation reaction requires your nuclei to be ~10x more concentrated than they are for snRNA-seq with only 5 uL available for your sample. Read more below for further insight into this issue.

What are the Common Multiome Sample Prep Problems?

Problem #1 – low input

Can we expect to sort more than 200,000 nuclei from your sample? If not your sample may suffer from the ‘low input’ problem

Normal Sample Prep Workflow:

The final resuspension buffer for snATAC or snATAC + Gene Expression is a Tris + MgCl2 buffer – but nuclei do not pellet in this buffer – they must be pelleted in a PBS+ BSA buffer.

Normal workflow: Sort into BSA buffer -> spin down -> remove supernatant -> resuspend in medium volume of ATAC buffer (100-200uls) -> count and load 5uls

Why is low input a problem?

With the normal workflow we have to resuspend in 100ul+ to dilute out the BSA buffer we need to pellet the nuclei – but we can only load 5ul of nuclei suspension.
If we sort 200,000 nuclei we can expect 5000+ nuclei to be recovered
If we only sort 25,000 we can expect a maximum of 800 cells captured – and at this cell number library quality is normally lower.

Possible Solution:

After sorting into a compatible buffer, remove supernatant, add diluted nuclei buffer (Tris + MgCl2) without disturbing pellet, and spin down again, then remove supernatant and resuspend in small volume
Caveats: This may cause loss and therefore will still be risky for samples with 50,000 nuclei or less

Summary of the Low Input Problem

ATAC + Gene Expression requires much larger numbers of input cells than scRNAseq in order to ensure a successful experiment.
200,000+ nuclei input (which really means closer to 300,000 cells as input if starting from a single cell suspension) should perform well and produce a good number of cells.
Cell numbers between 50,000 and 200,000 will likely perform well using the modified “low input” sample prep procedure

TL;DR: < 50,000 nuclei = big risk of a failed 10x multiome experiment

Problem #2- Difficult Cell Types

Does your sample contain large percentages of cell types that could be described as ‘bags of RNases’ (eg: Acinar Cells, Neutrophils)? If so, you may suffer from the ‘Difficult Cell Types’ problem.

Difficult Cell Types

During the lysis stage of the nuclei protocol, cells are lysed in a small volume of lysis buffer.
During lysis problematic cell types may release RNases which can degrade the RNA of all of the cells in the sample.
For this reason we capture very few neutrophils in scRNAseq because they degrade their own RNA in the droplet – but in snRNAseq this may degrade RNA of other cells.

Symptoms of this problem:

Degraded RNA can be seen in the cDNA trace of the bioanalyzer. Compare this trace of degraded snRNAseq cDNA vs a good sample:

Possible Solution: Depletion

The simplest potential solution to this problem is to deplete the problematic cell types.
Options for depletion are sorting with a granulocyte/ neutrophil marker or using a similar magnetic depletion kit

Summary of Difficult Cell Types Problem

RNase containing cell types may degrade the RNA of the whole sample during the nuclei isolation step.
Anecdotally, we have heard that pancreas is difficult to get successful snRNAseq data from and this is a potential reason why.
There are also suggestions that granulocytes can decrease the quality of the scATAC data (ICICLE/TEA-seq biorxiv paper)
This problem may vary by sample even within the same tissue type. Depletion seems to be the best option to prevent it.

TL;DR: High Numbers of Granulocytes/ “Difficult Cells” could cause Multiome RNAseq to fail

Problem # 3: Debris

Are there lots of weird non cell objects (large or small) in your single nuclei suspension? If so your sample may suffer from the debris problem.

Debris:

Problem: Debris can clog 10x Chromium
Solution: If debris is a problem and cannot be removed by filtering, a sorting strategy may need to be implemented ahead of the capture
Caveats: Many stains including PI and DAPI are not compatible with downstream ATAC when used to select for nuclei – can use 7-AAD

How to sort to remove debris using 7-AAD

Stain 1:100 with 7-AAD for ~ 5 minutes
Sort using the FL3 channel – sort into “ATAC Wash Dig” buffer (10mM Tris-HCL 7.4 , 10mM NaCl, 3mM MgCl, 1% BSA, .1% Tween, .1% Digitonin)
Gate for single nuclei ->

CSHL Single Cell Biology Core