TITLE: Optimising sample multiplexing oligos by flow cytometry
AUTHORS: Daniel V Brown

[DESCRIPTION]
Optmisation of sample multiplexing oligos by scRNA-Seq is costly and time consuming. A cheaper and faster method is to use a flow cytometry read-out with fluorescent detection oligonucleotides.
This method can also be used to mix samples with different fluorescently labelled oligos and investigate signal swapping.

[GUIDELINES]
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[STEPS]
SECTION: MULTI-Seq oligo preparation
2. Mix anchor and barcode strands in 1:1 molar ratio in PBS (without FBS or BSA at 2 µM concentration (10X stock).
SECTION: MULTI-Seq oligo preparation
2.1. This is 6uL 50uM anchor LMO, and 15uL 10uM barcode oligo to 129uL plain PBS.
SECTION: MULTI-Seq oligo preparation
2.2. Make one unique barcode solution per sample.
SECTION: MULTI-Seq oligo preparation
2.3. Total 25 µL per sample.
SECTION: MULTI-Seq oligo preparation
3. Make a 10X solution of the Co-Anchor in PBS.
SECTION: MULTI-Seq oligo preparation
3.1. Add 3uL 50uM co-anchor to 141uL plain PBS.
SECTION: MULTI-Seq oligo preparation
3.2. Add 6uL 100uM fluorescent detection oligo. For example:
Alexa 647 feature barcode 2 detection oligo.
SECTION: Prepare multiplexing reagent
1. For CellPlex and Hashtag antibody the reagent comes ready to use.
SECTION: Prepare multiplexing reagent
1.1. Prepare a dilution series for titration if desired
SECTION: Sample preparation
4. I use suspension cell lines to titrate cell multiplexing oligos so the sample preparation is easy.
SECTION: Sample preparation
4.1. Prepare a single-cell suspension of the sample to be tested.
SECTION: Sample preparation
4.2. Wash cells once in plain PBS without additives. I centrifuge suspension cell lines at 400xg.
Resuspend in PBS.
SECTION: Sample preparation
4.3. Count cells and transfer 100k to 1M cells (preferably 500k) into a 1.5mL tube for labelling
SECTION: Labelling samples with multiplexing oligos
5. This is largely based on original protocols but with the addition of a fluorescent secondary oligo.
SECTION: Labelling samples with multiplexing oligos
5.1. Resuspend cells in 180uL of plain PBS.
SECTION: Flow cytometry
6. Use a FACS analyser to compare the signal of labelled samples to a control sample where only the fluorescent detection oligo has been added.
If you have replicate labelled samples with different fluorescent detection oligos, you may combine them at this step.
SECTION: Flow cytometry
6.1. Add DAPI to a final concentration of 0.1ug/mL (1/100 stock tube).
SECTION: Labelling samples with multiplexing oligos
5.2. Add 20 µL 10X Anchor:Barcode solution and pipette gently to mix 10 – 15 times.
SECTION: Labelling samples with multiplexing oligos
5.3. Incubate on ice for 5 minutes.
SECTION: Labelling samples with multiplexing oligos
5.4. Add 20 µL Co-Anchor solution and pipette gently to mix.
SECTION: Labelling samples with multiplexing oligos
5.5. Incubate 5 minutes longer on ice.
SECTION: Labelling samples with multiplexing oligos
5.6. Add 1.5mL of 1% BSA in PBS (ice cold) to quench.
SECTION: Labelling samples with multiplexing oligos
5.7. Add 1.5mL of 1% BSA in PBS (ice cold) to quench.
SECTION: Labelling samples with multiplexing oligos
5.8. Add 1mL of 1% BSA in PBS (ice cold) to quench.
SECTION: Labelling samples with multiplexing oligos
5.9. Centrifuge cells at 4°C 400xg 5min.
SECTION: Labelling samples with multiplexing oligos
5.10. Resuspend cells in the remaining 100uL of supernatant then transfer to a new 1.5mL tube.
SECTION: Labelling samples with multiplexing oligos
5.11. Add 1.9mL 1% BSA in PBS and spin 5 minutes 400g at 4°C.
SECTION: Labelling samples with multiplexing oligos
5.12. Repeat wash 2 more times for a total of 3 washes.
SECTION: Labelling samples with multiplexing oligos
5.13. Resuspend cells in 500 µL of PBS + 1% BSA and transfer to 5mL polystyrene  FACS tube
SECTION: Flow cytometry
6.2. Gate for debris, single cells and viable cells.
SECTION: Flow cytometry
6.3. Aquire at least 10,000 viable cells per test.
SECTION: Flow cytometry
6.4. Analyse with relevant software.
SECTION: MULTI-Seq oligo preparation
3.3. It is highly recomended to label in at least duplicate. On these occasions I label the other replicate with a Alexa 594 detection oligo and mix immediately before FACS analysis.