Single cell RNA-seq trouble

[alekzs]

Hi folks,
I'm trying to set up a single-cell RNAseq platform for human primary cells, so far semi-successfull. Hopefully you can give me some feedback on how to optimize and fix the problems.

Here's what I do, somewhat following the Trombetta protocol http://onlinelibrary.wiley.com/doi/1...2s107/abstract :
1) FACS sort 1 cell/well into PCR plate with 10ul TCL buffer (+bMerc)
2) 2.2x RNA SPRI
3) RT with Maxima RNaseH-minus RT, 90min at 52C, then 10cycles 2min-50C, 2min-42C
4) WTA with KAPA HiFi Hotstart, 20 cycles
5) 2x 0.8x DNA SPRI

The good: I tested the protocol with Jurkat cells and got acceptable BioA traces for both a bulk control as well as 1cell/well samples (see attachment).

The bad: Using primary cells (human effector T cells or regulatory T cells, with the Tregs being my goal population) it went south. It works with >200 cells but I either get nothing out of my single cells or a weird pattern of cDNA distribution with 2-3 small bumps (I wouldn't even call them peaks).

What can I try to get it to work with 1 primary cell? And what causes this weird bumpy pattern?

Thanks
Alex

[lbcbci]

Single cell RNA-seq trouble

That would be due to significant lower RNA quantities from T cells than Jurkat cells

[omans]

Primary lymphocytes have a significantly lower level of mRNA than cultured cells.

I've found that scRNA-seq is very sensitive to sample handling. Are you using fresh or frozen T cells? How long is your sort?

Also, how many PCR cycles did you perform for each of these samples? For other lymphocyte types, I've needed to exceed 21 cycles to see a peak.

[alekzs]

I have tried increasing the PCR cycles up to 23 but still got this double-bump pattern.
The samples were frozen, I sort directly into lysis buffer (max 5min per plate), directly spin the plate down and then freeze on dry-ice and transfer to -80C until processing. For processing, I start out with bringing the RNA beads to room temp, RNAzap the work area, let the plates thaw on ice for 10min and then start the protocol.

[omans]

The double peak looks like degraded RNA. So there must be something that is causing the single primary cells, but not pooled sample or single jurkats to degrade. If you can identify that factor, you can probably fix it!

[Simone78]

I have tried increasing the PCR cycles up to 23 but still got this double-bump pattern.
The samples were frozen, I sort directly into lysis buffer (max 5min per plate), directly spin the plate down and then freeze on dry-ice and transfer to -80C until processing. For processing, I start out with bringing the RNA beads to room temp, RNAzap the work area, let the plates thaw on ice for 10min and then start the protocol.

I don´t have access to the paper you refer to but vaguely remember it.
I have some comments and suggestions:
- why do you sort in such a large volume of buffer. We generally sort in 2 ul (384w plates) or 4 ul (96w plates).
- use RNAse inhibitor at all stages, even during cell isolation. Is your FACS sorter clean...really clean? Always use negative controls in your test: 1 droplet of FACS buffer in a well with lysis buffer, to check that nothing gets amplified.
- use a dead/live marker. PI, CellTracker, whatever works (no Hoechst. it´s toxic and gives often issues)
- sorry my silly question but does "2.2X SPRI" mean? are you doing a bead purification before RT? I wouldn´t transfer and/or remove the content of the plate until after PCR, once you have enough DNA.
- I hope you have a mistake in the short protocol you posted and are not doing RT at 52 deg with Maxima H- but at 42 instead...
- It´s also not necessary to do 2 rounds of bead purification after PCR. If you are concerned with primer dimers use biotinylated primers and decrease the amount. But this of course, has no influence on the quality of your RNA of course...it just saves you time.

That said, frozen cells always have a much lower viability than fresh ones (a nice comparison here: PMID:28249587). For T cells we generally do 23-24 PCR cycles and you are doing the same, so no issue here. And yes, the "double bump" is a mix of degraded DNA (RNA) and leftover primers probably not removed after purification. Most likely you have a huge excess of unused primers after PCR.

Best,
Simone