Dec 29, 2023
Version 1
DoTA-seq V3 V.1
- 1UW Madison
Protocol Citation: freeman.lan 2023. DoTA-seq V3. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldzox7v5b/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 22, 2022
Last Modified: December 29, 2023
Protocol Integer ID: 69011
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.
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
MetaPolyzymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #MAC4L-5MG
Materials, Step 20
Pre-injection buffer 10mM HEPES pH 7.5 Pluronic 0.1%
In Materials and 2 steps
PBS 0.1% Tween20
In 2 steps
Cellbrite Fix 555BiotiumCatalog ##30088
Step 5
NN′-Bis(acryloyl)cystamineSanta Cruz BiotechnologyCatalog #sc-215506
Materials, Step 6
ddPCR Supermix for probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863024
Materials, Step 29
PerfluorooctanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #370533
In Materials and 2 steps
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
Step 35
High Sensitivity D1000 ScreenTapeAgilent TechnologiesCatalog #5067-5584
Step 41
Axygen® 0.2 mL Maxymum Recovery® Thin Wall PCR TubesCorningCatalog #PCR-02-L-C
Materials, Step 32
Biorad Evagreen Droplet OilBio-Rad LaboratoriesCatalog ##1864005
In Materials and 3 steps
PBS EDTA 1mM 0.1% w/v Tween 20
Step 18
HFE 7500 Perfluorinated Oil
Materials, Step 36
NEBNext Library Quant Kit for Illumina - 100 rxnsNew England BiolabsCatalog #E7630S
Materials, Step 42
Lysozyme from chicken egg whiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #L6876
In Materials, Materials, Step 20
PBS 0.1% Tween 20
Step 2
Isopropanol
Step 17
DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
Step 29
SYBR GreenThermo Fisher Scientific
Materials, Step 3
TCEP-HClGold BiotechnologyCatalog #TCEP
Materials, Step 38
AcrylamideP212121
Materials, Step 6
TEMED (Tetramethyl-ethulenediamine)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T9281
Materials, Step 10
Proteinase K solution, 20 mg ml − 1AmbionCatalog #AM2546
Materials, Step 23
10% SDSBio-Rad LaboratoriesCatalog #161-0146
Step 23
SPRIselect reagent kitBeckman CoulterCatalog #B23317
Step 40
DNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003
In Materials, Materials, Step 39
Acetone
Step 16
Ammonium persulfateCatalog #A3678
Materials, Step 6
PBS 2% Tween 20
Step 25
Preparing Cells
Preparing Cells
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
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)
Vortex Vigorously to Mix
Generate Gel Droplets
Generate Gel Droplets
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
Run the syringe pumps at 600uL/hr for the gel, and 1000uL/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 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 at 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 of AcetoneSigma Aldrich to the tube.
Wait 00:00:05 then remove with a pipette.
The gels should begin to flocculate and dehydrate.
5s
Add 1000 µL of IsopropanolSigma Aldrich
Wait 00:00:05 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.
5s
Resuspend in 1000 µL PBS EDTA 1mM 0.1% w/v Tween 20Sigma 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.
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, 00:00:15 each time
15s
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 01:00:00
1h
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 − 1Sigma AldrichCatalog #AM2546
100 µL 10% SDSSigma AldrichCatalog #161-0146
880 µL 1X PBS (Phosphate-buffered saline )Sigma Aldrich
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 PBS 2% Tween 20Sigma Aldrich
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.
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
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
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)Sigma AldrichCatalog #1863024
0.4 µL 50 micromolar (µM) P7 Primer
0.4 µL 50 micromolar (µM) P5 Primer with appropriate I5 index
0.2 µL Variable 10 micromolar (µM) DoTA-seq multiplex primer mix (10uM concentration per primer)
0.2 µL Variable 10 micromolar (µM) 16S DoTA-seq primers
0.5 µL Variable 1 picomolar (pM) Freshly diluted from 500pM stock Barcode Oligo
0.25 µL 500 millimolar (mM) Single use aliquots DTTSigma AldrichCatalog #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
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 an estimated 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.
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
Protocol
NAME
Loading Syringes to Inject into Microfluidics DeviceCREATED BY
freeman.lan
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
Run the syringe pumps at 200uL/hr for the gel and PCR mastermix, and 900uL/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
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
4h
PCR Cleanup
PCR Cleanup
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
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 28
Demaree B, Weisgerber D, Lan F, Abate AR. An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing.
https://doi.org/10.3791/57598