TITLE: DoTA-seq V3
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.

[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
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
2. Resuspend cells in 100 µL   

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)

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. Run the syringe pumps at 600uL/hr for the gel, and 1000uL/hr for the oil syringe. 
SECTION: Generate Gel Droplets
9. Collect gel droplets for 20 min  in a 1mL tube.

 

SECTION: Generate Gel Droplets
11. Add the Gel polymerization oil to the collected droplets 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
13. Use a pipette to remove the oil at 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.

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.
15. Pulse spin again and remove the oil in the bottom of the tube with a pipette.
16. Add 1000 µL of   to the tube.
Wait 5 s then remove with a pipette.
The gels should begin to flocculate and dehydrate.

17. Add 1000 µL of   
Wait 5 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.

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


SECTION: Lysing Bacteria
19. Wash gels 3 times in  1000 µL   (No Tween) by centrifugation at 500 x g, 15 s  each time
20. Make a Enzymatic Lysis Solution by adding:
20 mg   
100 µL 1 mg/mL   
900 µL   (No Tween)
21. Resuspend the gels in this lysis solution. Incubate at 37 °C for 60 min 

22. Wash the gels 3 times in 1000 µL   

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

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

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


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

SECTION: Barcoding the Cells
28. Load the gels into a syringe following the protocol described in this excellent visual protocol.
 

SECTION: Barcoding the Cells
29. 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
0.4 µL  50 micromolar (µM) P5 Primer with appropriate I5 index
0.2 µL 10 micromolar (µM) DoTA-seq multiplex primer mix (10uM concentration per primer)
0.2 µL  10 micromolar (µM) 16S DoTA-seq primers
0.5 µL 1 picomolar (pM) Barcode Oligo
0.25 µL 500 millimolar (mM)   

 

 

 

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


SECTION: Barcoding the Cells
31. Load 500 µL of   into a syringe following this protocol 
 


SECTION: Barcoding the Cells
32. Run the syringe pumps at 200uL/hr for the gel and PCR mastermix, and 900uL/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
SECTION: Barcoding the Cells
34. Thermocycle the PCR emulsion as follows:

95 °C  5 min

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

20 cycles of:
95 °C 30s 
72 °C 10s 
60 °C 90s 
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
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.  

SECTION: Generate Gel Droplets
10. Make 200 µL Gel Polymerization Oil - mix together in a tube:
195 µL   
5 µL