Sample requirements | SORT-seq

General

• Recommended: cell capture plate completely filled with single cells, 8 wells empty as a non-template control
• Minimal requirement: half a cell capture plate filled with single cells
• Plates: make sure the plates are well sealed with the provided aluminum seals, and make sure the plate ID (colored label) is still readable. Writing on the aluminum seal will not be processed by Single Cell Discoveries, we only use the plate ID and the information that is provided with the sample submission form.
• Shipment: in a plastic bag or cardboard Eppendorf box, plates taped together, plates with the right side up, on dry-ice. Please check our shipping section for more information.
  • Add to shipment: the sample (excel) sheet from the sample submission form.

FACS sorting

The essentials

There are a few things that are essential for a succesful sort:

1. The quality of your cells
   If the quality of the cells is not sufficient, the sequencing data will also be of low(er) quality. Therefore, we advise checking whether your cells survive the dissociation and the FACS sort before you plan your definitive sorting experiment. For more information on how to check the cell quality, please check our cell quality guide.
2. A proper gating strategy
   Without proper gating, you will end up with a suboptimal or even wrong population of cells. As a minimum, we advise selecting for live cells, by using a DAPI staining or other live/dead marker. It is also advisable to sort a few cells onto a microscopy slide or culture dish to check whether the morphology of the sorted particles fits your cell type of interest.
3. Proper calibration of the FACS sorter
   If the FACS machine is not appropriately aligned, cells will unfortunately not end up in the wells (correctly). Hence, it is essential to calibrate your FACS machine – every time – you start a sorting experiment. You can use the empty 384-wells plate, that was shipped with the cell capture plates, for the calibration. Seal the plate using one of the plate seals and sort FACS fluid/beads onto the foil at wells A1-3, H11-13 ór I11-13, and P22-24. Try to position the droplets in the middle of the wells. If you are sorting many plates during a day, it is advisable to calibrate a second time in between plates.
   
   Note: If using a BD FACS ARIA, the step size in one direction might be too large to center the stream properly. In this case, you can alter the voltage on the deflector plates to adjust the location of the droplets.
4. A clean FACS sorter
   This point probably goes without saying. If there is a contamination in your FACS machine, it will unfortunately also affect the sequencing data. Make sure you are using a well maintained and well-cleaned FACS machine when you are sorting into cell capture plates.

Plate layout

Many customers wonder in what fashion they should sort their cells; can you sort multiple cell types into one plate? Should you combine different control/experimental groups?

It is good to keep in mind that every experiment is different and that there is no standard answer to these questions. Some of our recommendations are:

• Always keep 8 wells in the right bottom corner of the plate empty
  These wells (O21-O24 and P21-P24) are your no-template control and could be important during data analysis.
• We usually recommend sorting one sample per plate
  The batch effects of the processing are usually minimal, especially when the biology is strong. The duration of the FACS sort is more of an influence than the processing. Usually, when sorting complex plate layouts, the cell sort takes longer, which can affect RNA quality and can create batch effects between sorted plates.
• Can I sort multiple cell types into one plate?
  Yes, especially when dealing with primary tissue, you may simply not have another choice. If you are combining cell cultures, keep in mind that some cells have significantly more RNA content and can overrule the other cells in the plate in terms of sequencing reads.

Recommended protocol for FACS sorting

1. Grab the cell capture plates from the -20°C approximately 15 min prior to the FACS sorting. Let them thaw on ice.
2. Make a single cell suspension according to your protocol of choice and pass the cell suspension through a FACS filter. Keep the cells on ice.
3. Spin the plates down (1-2 minutes, 1,500-2,000 x g) and keep them on ice.
4. Add a live/dead dye to your single-cell suspension 10 minutes prior to sorting. Vortex briefly and keep on ice.
5. Calibrate your FACS machine
6. Use block cooling if the FACS machine supports it
7. Select for live single cells during using proper FSC/SSC and live/dead gates (see figure below).
8. If your FACS machine supports it, store the index files that contain the FACS information for every single cell sorted into the plate. This could, for example, be important to adjust FACS gating later on, or if you want to overlay fluorescence information with transcriptome information from the same single cell.
9. Depending on how many cells you have, sorting one full plate should take somewhere between 7 and 15 minutes. Immediately move on with the following steps when a plate is done to prevent RNA degradation.
10. Cover the plate with a provided aluminum sealer. Avoid touching the plate bottom as much as possible while sealing the plate. Do try to seal the wells as properly as possible by using your hand or a plate sealing aid (but do not heat-seal).
11. After sealing, spin the plate for 2 min at 1,500-2,000 x g (preferably in a cooled centrifuge @ 4˚C)
12. Snap freeze each plate on dry ice. You can keep the plates on dry ice until the end of the sort. After the sorting experiment, all plates need to be stored at -80˚C until they are ready for shipment.

If you want, you can write additional info on the side of the plate, but make sure you remember which cells you sorted in which plate by keeping a record of the unique plate id (ABC-XY-001). SCD will only track the unique plate code upon receiving the plate.