TITLE: DoTA-seq V3.1
AUTHORS: freeman.lan

[DESCRIPTION]
This protocol describes the process of DoTA-seq generating a single cell sequencing library from a cell suspension. This workflow can be performed in two days, with the PCR step happening overnight. Before beginning this workflow make sure to have:

1. The necessary microfluidics devices prepared and ready to go
2. The multiplex DoTA-seq target primers validated to work together without generating large molecular weight primer dimers.

Please read the publication for further details.

[GUIDELINES]
Strongly recommend all pre-PCR steps (setting up reagents, washing gels) to be done in a PCR Clean hood. This has two purposes: 

1. Reduce PCR contamination of templates which can strongly effect single-cell PCR reactions.
2. Reduce dust contamination of reagents which can clog devices and cause failures.

[STEPS]
SECTION: Preparing Cells
1. Prepare a cell suspension by washing twice in 1mL of   by spinning down at 5000 x g, 1 min 

SECTION: Preparing Cells
2. Resuspend cells in 100 µL   

SECTION: Preparing Cells
3. Add 1 µL    10,000X dye to the cells to stain them

SECTION: Preparing Cells
4. Count cells using a hemacytometer using the SYBR signal, calculate concentration of the cell suspension.
SECTION: Preparing Cells
5. (Optional) Stain with   to get a cell membrane/wall stain
SECTION: Preparing Gel
6. Make  200 µL Hydrogel Precursor Solution - Mix together in a tube:
100 µL   monomer in water 25 Mass / % volume 
15 µL   in Methanol 5 Mass / % volume 
10 µL   10 Mass / % volume 
75 µL Cell suspension diluted in PBS (a total of 7e6 cells to achieve a final concentration of 3.5e7 cells/mL in the total solution)

Vortex Vigorously to Mix
SECTION: Generate Gel Droplets
7. Prepare and Load the Syringes with the gel sample and 600 µL   and connect to the microfluidic devices by following this protocol 
 



SECTION: Generate Gel Droplets
8. Connect the syringes to the inlets of the DoTA-seq Step 1 Microfluidics Device and Run the syringe pumps at 500uL/hr for the gel syringe, and 900uL/hr for the oil syringe. 


SECTION: Generate Gel Droplets
9. Collect gel droplets for 20 min  in a 1mL tube.

 

SECTION: Generate Gel Droplets
10. Make 200 µL Gel Polymerization Oil - mix together in a tube:
195 µL   
5 µL   
SECTION: Generate Gel Droplets
11. Add the Gel polymerization oil to the collected droplets, invert slowly 3 times to mix, and Incubate the tube containing droplets at 37 °C  for 10 min  to complete polymerization of the gel matrix.
 

SECTION: Breaking out gels from emulsion
12. Pulse spin the emulsion in a centrifuge to close pack the emulsion and drain the oil to the bottom of the tube.
SECTION: Breaking out gels from emulsion
13. Use a pipette to remove the oil from the bottom of the tube, leaving just the emulsion
SECTION: Breaking out gels from emulsion
14. Add 200 µL   to break the emulsion
Vortex, then Wait 1 min for the emulsion to break.

15. Pulse spin again and remove the oil in the bottom of the tube with a pipette.
17. Add 1000 µL of   to the tube.
Mix by inverting 5 times, Wait 10 s then remove with a pipette.
The gels should begin to flocculate and dehydrate.

18. Add 1000 µL of   
Mix by inverting 5 times, Wait 10 s then remove with a pipette.
The gels should dehydrate and become hard.

Note: Do not wait too long as it could cause the gels to irreversibly aggregate into clumps.

19. Resuspend in 1000 µL    
The gels can be stored at 4 °C  for several days without changing DoTA-seq results.
 


SECTION: Lysing Bacteria
20. Wash gels 3 times in  1000 µL   (No Tween) by centrifugation at 500 x g, 30 s  each time
21. Make a Enzymatic Lysis Solution by adding:
20 mg   
100 µL 1 mg/mL   
900 µL   (No Tween)
22. Resuspend the gels in this lysis solution. Incubate at 37 °C for 120 min 

23. Wash the gels 3 times in 1000 µL   

24. Make a SDS Lysis solution by adding:
20 µL 20 mg/mL   
100 µL   
880 µL  

25. Resuspend the gels in this SDS Lysis solution, incubate at 55 °C for 60 min 

26. Wash the gels three times in 1000 µL  
Note: Use 2% Tween 20, not 0.1% Tween
These gels can be stored at 4 °C in   for several days without impacting DoTA-seq results.
 




SECTION: Barcoding the Cells
27. Wash the gels three times in 1000 µL   
SECTION: Barcoding the Cells
28. Resuspend gels in 100 µL   

SECTION: Barcoding the Cells
29. Load the gels into a syringe following the protocol described in this excellent visual protocol.
 
Alternatively, for a simpler version, you can also use a P200 pipette to directly pipette the gel into a syringe backfilled with HFE7500.
SECTION: Barcoding the Cells
30. Generate a PCR Master Mix (This mix gives about ~10,000 cells per library - Scale up as required) 
25 µL   
0.4 µL 50 micromolar (µM) P7 Primer mixed with Barrev primer at 5 micromolar (µM) 
0.4 µL  50 micromolar (µM) P5 Primer with appropriate I5 index
0.2 µL 10 micromolar (µM) DoTA-seq multiplex primer mix (10-20nM final concentration per primer)
0.2 µL  10 micromolar (µM) 16S DoTA-seq primers (10-20nM final concentration)
0.5 µL 1 picomolar (pM) Barcode Oligo
0.25 µL 500 millimolar (mM)   

 

 

 

SECTION: Barcoding the Cells
31. Load the PCR mastermix into the syringe following this protocol 

Load 500 µL of   into a syringe following this protocol 
 


SECTION: Barcoding the Cells
32. Connect the syringes to the DoTA-seq Step 2 microfluidics device.

Run the syringe pumps at 200uL/hr for the gel and PCR mastermix, and 800uL/hr for the oil syringe.
Collect droplets in an    for 7 min for every 25 µL of PCR mastermix or until the PCR mastermix runs out.

SECTION: Barcoding the Cells
33. Use a pipette to remove the oil in the PCR tube, leaving just the emulsion layer (it's okay to have a little bit of oil remaining).
SECTION: Barcoding the Cells
34. Thermocycle the PCR emulsion as follows:

95 °C  5 min

40 cycles of:
95 °C 30s 
72 °C 10s 
60 °C 5 min 
72 °C 30s 

Final incubation of:
72 °C 10min
12 °C  Hold
All ramp times are at 1 °C  per second

SECTION: PCR Cleanup
35. Keep the emulsion on ice to prevent polymerase activity

Add 25 µL   to the emulsion
Vortex the emulsion to mix


SECTION: PCR Cleanup
36. Add 25 µL   to the emulsion
Add 25 µL   to the emulsion
Vortex the emulsion to mix
SECTION: PCR Cleanup
37. Wait 1 min , then pulse centrifuge to separate the PCR mix from the oil.

Note: If you do not see a clear separation of two clear phases and no emulsion remaining, repeat step 35.

Transfer the top aqueous phase to a new 1mL tube.

SECTION: PCR Cleanup
38. Add 20 µL 1 Molarity (M)   to the tube and vortex to completely decrosslink any remaining gels.
 


SECTION: PCR Cleanup
39. Clean up the PCR reaction using the kit.
Elute in 50 µL Elution Buffer.

SECTION: PCR Cleanup
40. Remove primer dimers and free barcodes using the   with 0.7X volume of beads.

SECTION: PCR Cleanup
41. Check the resulting library for primer dimers using 
 
 with a   
Other high sensitivity capillary electrophoresis methods will also work.

There should be minimal primer dimers on the trace. Below is an example of an acceptable trace.
 

SECTION: PCR Cleanup
42. Quantify the library using a qPCR library quantification kit such as   

 

SECTION: PCR Cleanup
43. Sequence the library on an Illumina sequencer using Custom Sequencing Primers listed here.  

16. Add 1000 µL of  to the tube.
Mix by inverting 5 times, Wait 10 s then remove with a pipette.