Dec 30, 2023
- 1UW Madison
Protocol Citation: freeman.lan 2023. DoTA-seq V3.1. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxx3ylpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 24, 2023
Last Modified: December 30, 2023
Protocol Integer ID: 86909
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:
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.
Materials
ddPCR Supermix for probes (no dUTP)BioRad SciencesCatalog #1863024
MetaPolyzymeSigma AldrichCatalog #MAC4L-5MG Lysozyme from chicken egg whiteSigma AldrichCatalog #L6876 HFE 7500 Perfluorinated Oil PerfluorooctanolSigma AldrichCatalog #370533 TCEP-HClGold BiotechnologyCatalog #TCEP NN′-Bis(acryloyl)cystamineSanta Cruz BiotechnologyCatalog #sc-215506 Ammonium persulfateCatalog #A3678 AcrylamideP212121 TEMED (Tetramethyl-ethulenediamine)Sigma-aldrichCatalog #T9281 Biorad Evagreen Droplet OilBioRad SciencesCatalog ##1864005 DNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003 DNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003 Axygen® 0.2 mL Maxymum Recovery® Thin Wall PCR TubesCorningCatalog #PCR-02-L-C NEBNext Library Quant Kit for Illumina - 100 rxnsNew England BiolabsCatalog #E7630S SYBR GreenThermo Fisher Scientific Proteinase K solution, 20 mg ml − 1AmbionCatalog #AM2546 Lysozyme from chicken egg whiteSigma AldrichCatalog #L6876 Pre-injection buffer 10mM HEPES pH 7.5 Pluronic 0.1%
Safety information
Unpolymerized Acrylamide is toxic, handle with care and dispose according to regulations
Protocol materials
SYBR GreenThermo Fisher Scientific
NN′-Bis(acryloyl)cystamineSanta Cruz BiotechnologyCatalog #sc-215506
Ammonium persulfateCatalog #A3678
TEMED (Tetramethyl-ethulenediamine)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T9281
MetaPolyzymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #MAC4L-5MG
Biorad Evagreen Droplet OilBio-Rad LaboratoriesCatalog ##1864005
DNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003
Proteinase K solution, 20 mg ml − 1AmbionCatalog #AM2546
Pre-injection buffer 10mM HEPES pH 7.5 Pluronic 0.1%
TCEP-HClGold BiotechnologyCatalog #TCEP
AcrylamideP212121
ddPCR Supermix for probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863024
Axygen® 0.2 mL Maxymum Recovery® Thin Wall PCR TubesCorningCatalog #PCR-02-L-C
NEBNext Library Quant Kit for Illumina - 100 rxnsNew England BiolabsCatalog #E7630S
Lysozyme from chicken egg whiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #L6876
Lysozyme from chicken egg whiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #L6876
PerfluorooctanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #370533
DNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003
HFE 7500 Perfluorinated Oil
PBS 0.1% Tween20
PBS 0.1% Tween 20
SYBR GreenThermo Fisher Scientific
Cellbrite Fix 555BiotiumCatalog ##30088
AcrylamideP212121
NN′-Bis(acryloyl)cystamineSanta Cruz BiotechnologyCatalog #sc-215506
Ammonium persulfateCatalog #A3678
Biorad Evagreen Droplet OilBio-Rad LaboratoriesCatalog ##1864005
TEMED (Tetramethyl-ethulenediamine)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T9281
Biorad Evagreen Droplet OilBio-Rad LaboratoriesCatalog ##1864005
PerfluorooctanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #370533
Acetone
Isopropanol
PBS EDTA 1mM Tween20 2% w/v
1X PBS (Phosphate-buffered saline )
Lysozyme from chicken egg whiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #L6876
MetaPolyzymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #MAC4L-5MG
1X PBS (Phosphate-buffered saline )
PBS 0.1% Tween20
Proteinase K solution, 20 mg ml − 1AmbionCatalog #AM2546
10% SDSBio-Rad LaboratoriesCatalog #161-0146
TE Buffer (Tris 10mM EDTA 1mM pH 8)
TE 2% Tween-20
TE 2% Tween-20
Pre-injection buffer 10mM HEPES pH 7.5 Pluronic 0.1%
Pre-injection buffer 10mM HEPES pH 7.5 Pluronic 0.1%
ddPCR Supermix for probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863024
DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
Biorad Evagreen Droplet OilBio-Rad LaboratoriesCatalog ##1864005
Axygen® 0.2 mL Maxymum Recovery® Thin Wall PCR TubesCorningCatalog #PCR-02-L-C
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
HFE 7500 Perfluorinated Oil
PerfluorooctanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #370533
TCEP-HClGold BiotechnologyCatalog #TCEP
DNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003
SPRIselect reagent kitBeckman CoulterCatalog #B23317
High Sensitivity D1000 ScreenTapeAgilent TechnologiesCatalog #5067-5584
NEBNext Library Quant Kit for Illumina - 100 rxnsNew England BiolabsCatalog #E7630S
HFE 7500 Perfluorinated Oil
Preparing Cells
Preparing Cells
10m
10m
Prepare a cell suspension by washing twice in 1mL of PBS 0.1% Tween20Sigma Aldrich by spinning down at 5000 x g, 00:01:00
Resuspend cells in 100 µL PBS 0.1% Tween 20Sigma Aldrich
Add 1 µL SYBR GreenSigma Aldrich 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 Cellbrite Fix 555Sigma AldrichCatalog ##30088 to get a cell membrane/wall stain
Preparing Gel
Preparing Gel
30m
30m
Make 200 µL Hydrogel Precursor Solution - Mix together in a tube:
100 µL AcrylamideSigma Aldrich monomer in water 25 Mass / % volume
15 µL NN′-Bis(acryloyl)cystamineSigma AldrichCatalog #sc-215506 in Methanol 5 Mass / % volume
10 µL Ammonium persulfateSigma AldrichCatalog #A3678 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
Generate Gel Droplets
Generate Gel Droplets
10m
10m
Prepare and Load the Syringes with the gel sample and 600 µL Biorad Evagreen Droplet OilSigma AldrichCatalog ##1864005 and connect to the microfluidic devices by following this protocol
Protocol
NAME
Loading Syringes to Inject into Microfluidics DeviceCREATED BY
freeman.lan
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 00:20:00 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.
20m
Make 200 µL Gel Polymerization Oil - mix together in a tube:
195 µL Biorad Evagreen Droplet OilSigma AldrichCatalog ##1864005
5 µL TEMED (Tetramethyl-ethulenediamine)Sigma AldrichCatalog #T9281
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 00:10:00 to complete polymerization of the gel matrix.
Note
You can now look at the emulsion under the microscope using Countess slidesThermo Fisher ScientificCatalog #C10228 to determine the encapsulation ratio of your cells. SYBRGreen and CF555 signal should be concordant and correspond to cells.
10m
Breaking out gels from emulsion
Breaking out gels from emulsion
Pulse spin the emulsion in a centrifuge to close pack the emulsion and drain the oil to the bottom of the tube.
Use a pipette to remove the oil from the bottom of the tube, leaving just the emulsion
Add 200 µL PerfluorooctanolSigma AldrichCatalog #370533 to break the emulsion
Vortex, then Wait 00:01:00 for the emulsion to break.
1m
Pulse spin again and remove the oil in the bottom of the tube with a pipette.
Add 1000 µL ofHFE 7500 Perfluorinated OilContributed by users to the tube.
Mix by inverting 5 times, Wait 00:00:10 then remove with a pipette.
10s
Add 1000 µL of AcetoneSigma Aldrich to the tube.
Mix by inverting 5 times, Wait 00:00:10 then remove with a pipette.
The gels should begin to flocculate and dehydrate.
10s
Add 1000 µL of IsopropanolSigma Aldrich
Mix by inverting 5 times, Wait 00:00:10 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.
10s
Resuspend in 1000 µL PBS EDTA 1mM Tween20 2% w/vSigma Aldrich
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 slidesThermo Fisher ScientificCatalog #C10228 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
Lysing Bacteria
Wash gels 3 times in 1000 µL 1X PBS (Phosphate-buffered saline )Sigma Aldrich (No Tween) by centrifugation at 500 x g, 00:00:30 each time
30s
Make a Enzymatic Lysis Solution by adding:
20 mg Lysozyme from chicken egg whiteSigma AldrichCatalog #L6876
100 µL 1 mg/mL MetaPolyzymeSigma AldrichCatalog #MAC4L-5MG
900 µL 1X PBS (Phosphate-buffered saline )Sigma Aldrich (No Tween)
Resuspend the gels in this lysis solution. Incubate at 37 °C for 02:00:00
2h
Wash the gels 3 times in 1000 µL PBS 0.1% Tween20Sigma Aldrich
Make a SDS Lysis solution by adding:
20 µL 20 mg/mL Proteinase K solution, 20 mg ml − 1AmbionCatalog #AM2546
100 µL 10% SDSBio-Rad LaboratoriesCatalog #161-0146
880 µL TE Buffer (Tris 10mM EDTA 1mM pH 8)
Resuspend the gels in this SDS Lysis solution, incubate at 55 °C for 01:00:00
1h
Wash the gels three times in 1000 µL TE 2% Tween-20
Note: Use 2% Tween 20, not 0.1% Tween
These gels can be stored at 4 °C in TE 2% Tween-20 Contributed by users for several days without impacting DoTA-seq results.
Note
You can now look at the gels under the microscope using Countess slidesThermo Fisher ScientificCatalog #C10228 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
Barcoding the Cells
7m
7m
Wash the gels three times in 1000 µL Pre-injection buffer 10mM HEPES pH 7.5 Pluronic 0.1%Sigma Aldrich
Resuspend gels in 100 µL Pre-injection buffer 10mM HEPES pH 7.5 Pluronic 0.1%Sigma Aldrich
Load the gels into a syringe following the protocol described in this excellent visual protocol.
CITATION
Alternatively, for a simpler version, you can also use a P200 pipette to directly pipette the gel into a syringe backfilled with HFE7500.
Generate a PCR Master Mix (This mix gives about ~10,000 cells per library - Scale up as required)
25 µL ddPCR Supermix for probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863024
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 Variable 10 micromolar (µM) DoTA-seq multiplex primer mix (10-20nM final concentration per primer)
0.2 µL Variable 10 micromolar (µM) 16S DoTA-seq primers (10-20nM final concentration)
0.5 µL Variable 1 picomolar (pM) Freshly diluted from 500pM stock Barcode Oligo
0.25 µL 500 millimolar (mM) Single use aliquots DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
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.
20m
Load the PCR mastermix into the syringe following this protocol
Load 500 µL of Biorad Evagreen Droplet OilSigma AldrichCatalog ##1864005 into a syringe following this protocol
Protocol
NAME
Loading Syringes to Inject into Microfluidics DeviceCREATED BY
freeman.lan
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 Axygen® 0.2 mL Maxymum Recovery® Thin Wall PCR TubesSigma AldrichCatalog #PCR-02-L-C for 00:07:00 for every 25 µL of PCR mastermix or until the PCR mastermix runs out.
7m
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).
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
4h
PCR Cleanup
PCR Cleanup
1m
1m
Keep the emulsion on ice to prevent polymerase activity
Add 25 µL EDTA (0.5 M), pH 8.0Sigma AldrichCatalog #AM9260G to the emulsion
Vortex the emulsion to mix
Add 25 µL HFE 7500 Perfluorinated OilSigma Aldrich to the emulsion
Add 25 µL PerfluorooctanolSigma AldrichCatalog #370533 to the emulsion
Vortex the emulsion to mix
Wait 00:01:00 , 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.
1m
Add 20 µL 1 Molarity (M) TCEP-HClSigma AldrichCatalog #TCEP 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.
Clean up the PCR reaction using theDNA Clean & Concentrator™-5Sigma AldrichCatalog #D4003 kit.
Elute in 50 µL Elution Buffer.
10m
Remove primer dimers and free barcodes using the SPRIselect reagent kitSigma AldrichCatalog #B23317 with 0.7X volume of beads.
10m
Check the resulting library for primer dimers using
Equipment
TapeStation
NAME
Agilent
BRAND
G2991AA
SKU
LINK
with a High Sensitivity D1000 ScreenTapeSigma AldrichCatalog #5067-5584
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.
10m
Quantify the library using a qPCR library quantification kit such as NEBNext Library Quant Kit for Illumina - 100 rxnsSigma AldrichCatalog #E7630S
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!
1h
Sequence the library on an Illumina sequencer using Custom Sequencing Primers listed here.
DoTA-seq-Oligo-Sequences.xlsx
DoTA-seq-Oligo-Sequences.xlsx Citations
Step 29
Demaree B, Weisgerber D, Lan F, Abate AR. An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing.
https://doi.org/10.3791/57598