DoTA-seq V3.1

freeman.lan

Published: 2023-12-30 DOI: 10.17504/protocols.io.81wgbxx3ylpk/v1

Abstract

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:

The necessary microfluidics devices prepared and ready to go
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

Preparing Cells

Prepare a cell suspension by washing twice in 1mL of by spinning down at 5000x g

Resuspend cells in 100µL

Add 1µL 10,000X dye to the cells to stain them

Count cells using a hemacytometer using the SYBR signal, calculate concentration of the cell suspension.

(Optional) Stain with to get a cell membrane/wall stain

Preparing Gel

Make 200µL Hydrogel Precursor Solution - Mix together in a tube:

100µL monomer in water 25Mass / % volume

15µL in Methanol 5Mass / % volume

10µL 10Mass / % volume

75µL 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

Generate Gel Droplets

Prepare and Load the Syringes with the gel sample and 600µL and connect to the microfluidic devices by following this protocol

Loading Syringes to Inject into Microfluidics Device

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.

Collect gel droplets for 0h 20m 0s in a 1mL tube.

Note

Sometimes the initial droplet formation produces polydisperse droplets. In this case, wait 2 min for the bad emulsion to leave the outlet tubing into a waste tube, then begin collecting in the collection tube.

10.

Make 200µL Gel Polymerization Oil - mix together in a tube:

195µL

5µL

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 0h 10m 0s to complete polymerization of the gel matrix.

Note

You can now look at the emulsion under the microscope using Countess slides to determine the encapsulation ratio of your cells. SYBRGreen and CF555 signal should be concordant and correspond to cells.

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.

13.

Use a pipette to remove the oil from the bottom of the tube, leaving just the emulsion

14.

Add 200µL to break the emulsion

Vortex, then Wait 0h 1m 0s for the emulsion to break.

15.

Pulse spin again and remove the oil in the bottom of the tube with a pipette.

16.

Add 1000µL of to the tube.

Mix by inverting 5 times, Wait 0h 0m 10s then remove with a pipette.

17.

Add 1000µL of to the tube.

Mix by inverting 5 times, Wait 0h 0m 10s then remove with a pipette.

The gels should begin to flocculate and dehydrate.

18.

Add 1000µL of

Mix by inverting 5 times, Wait 0h 0m 10s 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 1000µL

The gels can be stored at 4°C for several days without changing DoTA-seq results.

Note

You can now look at the gels under the microscope using Countess slides to determine the encapsulation ratio of your cells. For SYBR Staining, you should first wash a small aliquot in PBS 2% Tween to remove background SYBR+ oil droplets before visualization on the microscope.You should see some loss in CF555 signal as the acetone and alcohol wash removes some bacterial membranes.

Lysing Bacteria

20.

Wash gels 3 times in 1000µL (No Tween) by centrifugation at 500x g each time

21.

Make a Enzymatic Lysis Solution by adding:

20mg

100µL 1mg/mL

900µL (No Tween)

22.

Resuspend the gels in this lysis solution. Incubate at 37°C for 2h 0m 0s

23.

Wash the gels 3 times in 1000µL

24.

Make a SDS Lysis solution by adding:

20µL 20mg/mL

100µL

880µL

25.

Resuspend the gels in this SDS Lysis solution, incubate at 55°C for 1h 0m 0s

26.

Wash the gels three times in 1000µL

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.

Note

You can now look at the gels under the microscope using Countess slides to determine the encapsulation ratio and lysis efficiency of your cells. It is advised to restain with SYBR and CF555 to get best signal. Lysed cells should exhibit SYBR signal but no CF555 Signal.

Barcoding the Cells

27.

Wash the gels three times in 1000µL

28.

Resuspend gels in 100µL

29.

Load the gels into a syringe following the protocol described in this excellent visual protocol.

Citation

Demaree B, Weisgerber D, Lan F, Abate AR 2018 An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing. Journal of visualized experiments : JoVE https://doi.org/10.3791/57598

Alternatively, for a simpler version, you can also use a P200 pipette to directly pipette the gel into a syringe backfilled with HFE7500.

30.

Generate a PCR Master Mix (This mix gives about ~10,000 cells per library - Scale up as required)

25µL

0.4µL 50micromolar (µM) P7 Primer mixed with Barrev primer at 5micromolar (µM)

0.4µL 50micromolar (µM) P5 Primer with appropriate I5 index

0.2µL 10micromolar (µM) DoTA-seq multiplex primer mix ( 10-20nM final concentration per prime r)

0.2µL 10micromolar (µM) 16S DoTA-seq primers ( 10-20nM final concentration )

0.5µL 1picomolar (pM) Barcode Oligo

0.25µL 500millimolar (mM)

Note

The ratio of 16S to DoTA-seq target primers mix can be varied depending on the relative amplification efficiencies. The best way to determine is to start from equal concentrations, then adjust based on the sequencing results (do most cells contain more 16S reads than target reads?)

Note

IMPORTANT - BARCODE CONCENTRATIONS MAY NEED TO BE MEASURED Typically, 0.5uL of 1pM barcode will give approximately 1 barcode for every 10 droplets. However, it is best to measure the barcode encapsulation rate by making PCR droplets containing the barcodes at the expected dilution and P7 and Barrev primers targeting the barcode for amplification. Visualize the resulting PCR emulsion using SYBRgreen staining under the microscope to obtain the real encapsulation ratio. Typically, the real barcode concentration can be ~5 fold off from the expected concentration based on manufacturer's labelling.

Note

Barcode oligos should always be freshly diluted from 500pM to 1pM before use, as we have found gradual loss of barcodes over time in a 1pM solution.

31.

Load the PCR mastermix into the syringe following this protocol

Load 500µL of into a syringe following this protocol

Loading Syringes to Inject into Microfluidics Device

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 0h 7m 0s for every 25µL of PCR mastermix or until the PCR mastermix runs out.

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).

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

PCR Cleanup

35.

Keep the emulsion on ice to prevent polymerase activity

Add 25µL to the emulsion

Vortex the emulsion to mix

36.

Add 25µL to the emulsion

Vortex the emulsion to mix

37.

Wait 0h 1m 0s , 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.

38.

Add 20µL 1Molarity (M) to the tube and vortex to completely decrosslink any remaining gels.

Note

You should be unable to obtain any "jellyish" substance by centrifugation! If there is any jellyish substance left it is not fully de-crosslinked. ADD MORE TCEP.

39.

Clean up the PCR reaction using thekit.

Elute in 50µL Elution Buffer.

40.

Remove primer dimers and free barcodes using the with 0.7X volume of beads.

41.

Check the resulting library for primer dimers using

Equipment

Value	Label
TapeStation	NAME
Agilent	BRAND
G2991AA	SKU

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.

Example of an acceptable Tapestation trace.

42.

Quantify the library using a qPCR library quantification kit such as

Note

Note that you must use a PCR based library quantification kit as not all amplicons contain all the adaptors for sequencing and therefore will throw off sequence non-specific forms of quantification!

43.

Sequence the library on an Illumina sequencer using Custom Sequencing Primers listed here.DoTA-seq-Oligo-Sequences.xlsx

DoTA-seq V3.1

Abstract

Steps

Preparing Cells

Preparing Gel

Generate Gel Droplets

Breaking out gels from emulsion

Lysing Bacteria

Barcoding the Cells

PCR Cleanup

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