- How to be a happy sorter
- Sorting strategies
- OPTION A: sorting into media
- OPTION B: sorting into lysis buffer
How to be a happy sorter
Sorting and handling of samples prior to RNA/cDNA/library prep, is the most important factor that will influence your ability to make high quality sequence-ready libraries. The suggestions below come from years of trial-and-error from labs that we've collaborated with, so ignore at your own peril!
- Always keep your cells on ice during the sort (I know, seems obvious, but ya never know)
- Always check viability after your sort. Good ol' Trypan Blue and a hemocytometer is fine.
- Always confirm your sort purity by staining and running some of your sorted cells back through a cytometer. It's worth it, trust me.
- Consider double or even triple sorting your cells (I know, wild right?!). 'Well that's too damn expensive!', you say? Not nearly as expensive as moving ahead with sequencing and analysis, only to find out that all of your top genes are from a contaminating cell population (see here). Double/triple sorts are particularly important if you're comparing very similar cell populations, like the same subset of T cells from WT vs KO mice. In these situations, even subtle differences in the number or type of contaminating cells between your groups can totally dominate
- If you're expecting a lot of time to pass between getting your first and last sample sorted, you'll want to think about how you can either prevent or control for potential impacts this could have on cell viability and/or gene expression. Here are a few options:
- See point # 1 above
- Team up with someone in your lab who can process each sample to extract RNA immediately after the sort
- Don't sort by group/condition/treatment, instead mix it up. This will help avoid a batch effect that is confounded with the variable of interest.
OPTION A: sorting into media
We recommend Option A if you plan to sort > 3000 cells. Following sorting, RNA is extracted and used as input for cDNA generation.
There is a lot of variability in terms of how sensitive cells are to the sorting process – naïve and effector T cells do great, others (like Treg cells) are way more fragile. For these more challenging cell types, you should plan to sort 3-4 times more cells than you need.
- Always use the 100 uM nozzle on the sorter (rather than 70 uM) and sort at low speed (~5-7K events/sec). This will be more gentle on the cells
- Sort into Lo-bind 96-well plates to prevent cells from sticking to sides (Lo-bind gives tighter pellets after spin down)
- Sort into complete media with 50% serum (RPMI with hepes, Pen/Strep, NEAA, L-glut, Na-Pyruvate, 2ME, etc.)
Check the purity of your sort by taking 5-10 ul and running back throught the cytometer.
- Keep everything cold: cells, sort chamber, collection block, collection tubes, etc.
- Minimize time after sort to lysis. If sorting many samples, spin down in batches – don’t wait until the end if possible.
- Spin down cells. ~10,000 cells usually produce a visible pellet. Use a pipette tip to carefully draw off supe.
- lyse cells by adding 300 ul of buffer RLT (from Qiagen RNeasy kit) with 2-ME added fresh. Vortex to lyse cells. Spin down hard.
- Flash freeze tube on dry ice and transfer to a prechilled box at -80C.
OPTION B: sorting into lysis buffer
We recommend Option B if you plan to sort < 3000 cells. You will sort directly into lysis buffer and skip RNA extraction entirely, using the crude cell lysate for input directly into the cDNA reaction.
The 10x lysis buffer used in the next step contains detergent, so avoid creating bubbles when pipetting.
- Prepare 10x reaction buffer from the SMART-Seq HT kit by mixing 19 uL of 10x Lysis Buffer with 1 uL of RNase inhibitor. This is enough for ~20 samples. Scale up as needed, but be sure to maintain 19:1 ratio of lysis buffer to RNase inhibitor
- Prepare 1x reaction buffer by mixing 9.5 uL nuclease-free water with 1 uL of 10x reaction buffer. This is enough to sort one sample, so be sure to scale up as needed.
- Add 5ul of 1x reaction buffer to collection eppendorf tube (or one well of a 96-well plate) and sort directly into this tube or well.
- Immediately after sample is sorted, add an additional 5.5 uL of 1x reaction buffer to tube.
- Store at -80C until ready to begin cDNA synthesis. You can proceed directly from this cell lysate to cDNA preparation with no intermediate RNA isolation step.