Jun 21, 2022
Version 3
Direct nuclear tagmentation and RNA-sequencing (DNTR-seq) V.3
This protocol is a draft, published without a DOI.
- Vasilios Zachariadis1,
- Huaitao Cheng1,
- Nathanael Andrews1,
- Martin Enge1
- 1Karolinska Institutet
- Enge lab
Protocol Citation: Vasilios Zachariadis, Huaitao Cheng, Nathanael Andrews, Martin Enge 2022. Direct nuclear tagmentation and RNA-sequencing (DNTR-seq). protocols.io https://protocols.io/view/direct-nuclear-tagmentation-and-rna-sequencing-dnt-b65yrg7wVersion created by Vasilios Zachariadis
Manuscript citation:
A highly scalable method for joint whole genome sequencing and gene expression profiling of single cells bioRxiv 2020.03.04.976530; doi: https://doi.org/10.1101/2020.03.04.976530
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: April 05, 2022
Last Modified: June 21, 2022
Protocol Integer ID: 60312
Keywords: single-cell, scRNA-seq, scWGS,
Abstract
Understanding how genetic variation alters gene expression - how genotype affects phenotype - is a central challenge in biology. To address this question in complex cell mixtures, we developed Direct Nuclear Tagmentation and RNA-sequencing (DNTR-seq), which enables whole genome and mRNA sequencing jointly in single cells.
Guidelines
Oligonucleotides (all ordered from IDT using Standard desalting, except barcodes ordered in solution/plates)
Oligo-dT: AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT(N1:34333300)(N2:25252525)
IS_PCR: 5′-AAGCAGTGGTATCAACGCAGAGT-3′
TSO: 5′-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′
ME-A: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3'
ME-B: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'
ME-Rev: 5'-/5Phos/CTGTCTCTTATACACATCT-3'
Illumina-compatible barcodes used (Sxxx/Nxxx series, n=784) are available as a supplementary table in the manuscript.
Materials
MATERIALS
HotStart ReadyMix (KAPA HiFi PCR kit)Kapa BiosystemsCatalog #KK2601
Proteinase KThermo Fisher ScientificCatalog #EO0491
Tween-20Sigma-aldrichCatalog #P-7949
psfTn5addgeneCatalog #79107
10% SDS solutionTeknovaCatalog #S0287
SMARTScribe Reverse TranscriptaseTakarabioCatalog #634888
Magnesium chloride solution for molecular biology (1.00 M)Sigma – AldrichCatalog #M1028
Ice
Triton X-100 SigmaCatalog #93426
Microseal® ‘F’ Foil BioRad SciencesCatalog #MSF-1001
dNTP Mix (dATP, dCTP, dGTP, and dTTP, each at 10mM) Thermo Fisher ScientificCatalog #R0192
KAPA HiFi PCR kit with dNTPsFisher ScientificCatalog #NC0142652
Betaine 5MSigma AldrichCatalog #B0300
Dry ice
UltraPure™ DNase/RNase-Free Distilled WaterThermo FisherCatalog #10977035
ERCC RNA Spike-In MixThermo FisherCatalog #4456740
USB Dithiothreitol (DTT), 0.1M SolutionThermo FisherCatalog #707265ML
Sera-Mag Speed BeadsGe HealthcareCatalog #65152105050250
RNase InhibitorTakaraCatalog #2313A
Hard-Shell® 384-Well PCR Plates thin wall skirtedBioRad SciencesCatalog #HSP3801
Before start
Bleach clean environment - to avoid DNA contamination. And RNase away or similar to avoid degraded RNAs. Prepare solutions in a strictly pre-PCR environment. Keep plates and reagents on ice unless otherwise noted.
Prepare lysis buffer plates for cell sorting
Prepare lysis buffer plates for cell sorting
Prepare lysis buffer mix
NOTE: Reagents are prepared on ice, working quickly. ERCC is stored in single-use aliquots at -80 °C , thawed on ice and added last.
| A | B | C | D | |
| Reagent | Reaction conc. | µL per reaction | 384w plate (400x) | |
| Nuclease free H2O | - | 1.965 | 786 | |
| RNase Inhibitor (40u/µL) | 1 unit/µL | 0.075 | 30 | |
| ERCC (1:1 200 000) | - | 0.075 | 30 | |
| Triton-X100 (10% solution) | 0.2% | 0.06 | 24 | |
| dNTP (10mM each) | 2.5mM/each | 0.75 | 300 | |
| Oligo-dT (100µM) | 2.5µM | 0.075 | 30 | |
| To dispense | 3 | 1200 |
Add 3 µL lysis buffer mix to each well. Cover with appropriate lids. Spin down.
Snap freeze on dry ice. Store until use at -80 °C
Sort single-cells
Sort single-cells
Sort single cells into 3 µL lysis buffer mix
Immediately seal with appropriate seals (approved for -80C > 100C) and centrifuge at 2000 x g, 4°C, 00:05:00
Snap freeze on dry ice. Store until use at -80 °C
Separation of nuclear and cytosolic fractions
Separation of nuclear and cytosolic fractions
Thaw plate on ice.
Centrifuge at 500 x g, 4°C, 00:05:00 .
Keep on ice.
Transfer 2 µL from each well of the sorted plate into an empty 384-well plate. Use a low flow rate (2mm/s) and an aspiration height of 0.9mm above the bottom.
Note
NOTE: We use the Eppendorf EpMotion 5073m benchtop liquid handler. We have succesfully used other solutions, including the Hamilton STARlet, a semi-manual Gilson Platemaster 96-well pipette, and even manual 8-channel pipettes.
Spin down and freeze nuclear fraction at -20 °C to aid complete lysis.
If proceeding with cDNA protocol --> step 12.
If proceeding with DNA protocol (step 6): spin down and snap freeze cytosolic fraction on dry ice and store at -80 °C
Note
NOTE: We will typically proceed with cDNA synthesis, unless experimental design dictates otherwise, to avoid an additional freeze-thaw cycle for mRNAs in the cytosolic fraction.
Single-cell genomic libraries
Single-cell genomic libraries
Using plate with nuclear fraction, with remaining volume 1µl/well.
Proteinase K treatment
1. Dilute Proteinase K (stock 20mg/ml) to 0.2mg/ml by 30mM Tris-HCl pH8.0
2. Add 2 µL diluted Proteinase K (0.2mg/ml) to each well. Makes 0.13mg/ml reaction concentration.
3. Incubate in thermocycler at:
- 50 °C 01:00:00
- 80 °C 00:30:00
- 4 °C hold
Tn5 digestion
Tn5 is produced from psfTn5 (Addgene #79107), purified to ~3mg/ml and assembled with Illumina Tn5 adapters (see oligos) as in Picelli et al, 2014.
CITATION
Prepare 2X Tn5 Buffer. Keep assembled Tn5 enzyme (Picelli et al, 2014) on ice block and add last.
| A | B | C | D | |
| Reagent | Reaction conc | µL per reaction | 384w plate (420x) | |
| 5X TAPS-PEG (50mM TAPS, 25mM MgCl2, 40% PEG-8000) | 10mM TAPS 5mM MgCl2 8% PEG-8000 | 1.6 | 672 | |
| psfTn5, loaded with 50µM MEDS-A/B | 0.1 | 42 | ||
| Nuclease free H2O | 3.3 | 1386 | ||
| To dispense | 5 | 2100 |
Add 5 µL per well. Vortex and spin down plate.
Note
NOTE: Buffer contains PEG, which is viscous. 5X TAPS-PEG buffer should be allowed to assume room temperature before dispensing to allow proper mixing.
Incubate in thermocycler: 55 °C 00:10:00
Remove immediately and stop reaction by adding 2 µL per well of 0.2% SDS.
Vortex, spin down and incubate 00:10:00 at 55 °C
PCR amplification and barcoding
1. Prepare PCR master-mix
| A | B | C | D | |
| Reagent | Reaction conc. | µl per reaction | 384w plate | |
| Nuclease free H2O | - | 2.9 | 1218 | |
| KAPA HiFi Buffer (5X) | 1X | 3.9 | 1638 | |
| dNTP (10mM/each) | 0.3mM/each | 0.6 | 252 | |
| KAPA enzyme (1u/µl) | 0.02u/µl | 0.4 | 168 | |
| Tween-20 (10%) | 0.1% | 0.2 | 84 | |
| To dispense | 8 | 3360 | ||
2. Dispense 8 µL per well
3. Add primers/barcodes 1.5 µL per well (from 384-well index plates, with 3.75µM/each forward/reverse primers; see oligos). Total reaction volume is now 19.5µl (10µl sample + 9.5µl PCR mix and primers).
4. Vortex plate, spin down and incubate in thermocycler with the following program:
| Step | Temperature | Time | Cycles | |
| Gap fill | 72ºC | 3 min | 1x | |
| First denature | 95ºC | 30 sec | 1x | |
| Denature | 95ºC | 15 sec | 18x | |
| Anneal | 67ºC | 30 sec | ||
| Extend | 72ºC | 45 sec | ||
| Final extension | 72ºC | 4 min | 1x | |
| 4-10ºC | hold |
Pool 3 µL from each well into a 1.5mL Eppendorf tube.
Library cleanup
We prepare SPRI-beads in 20% PEG-8000 solution as in: https://openwetware.org/wiki/SPRI_bead_mix#Ingredients_for_50_mL_2
(optional) Take an aliquot of your pool (300µl)
1. Add 0.9X SPRI-beads in 20% PEG solution. Incubate for 00:05:00 Room temperature
2. Place on magnetic rack 00:03:00
3. Remove supernatant
4. Add 1 volume 80% EtOH (fresh). Incubate for 00:00:30
5. Remove supernatant
6. Repeat EtOH wash
7. Air dry for 00:10:00 - - 00:15:00
8. Re-suspend beads thoroughly in 100 µL EB or TE buffer
9. Repeat cleanup (from step 1-7) and elute in 30 µL EB or TE buffer
(optional) Quality control of DNA libraries
Using Agilent HS 5000 DNA chips (or equivalent)
Pooled (and dliuted) DNA-library from 384-well plate.
This library was sequenced on a NextSeq 550 loading 2.5pM based on a peak of 334bp.
Sequencing was paired-end 37bp, 8bp dual index.
Reverse transcription and cDNA amplification
Reverse transcription and cDNA amplification
Following step 4, cytosolic/RNA fraction plate contains 2µl solution per well.
Primer annealing
Thaw plate. Spin down. Incubate in thermocycler at 72 °C for00:03:00 . Remove to ice immediately.
Prepare RT master-mix
| A | B | C | D | |
| Reagent | Reaction conc. | µl per reaction | 384w plate (420x) | |
| Maxima H Minus RT (200/µl) | 2u/µl | 0.05 | 21 | |
| RNase Inhibitor (40u/µl) | 1.66u/µl | 0.125 | 52.5 | |
| 5X First Strand buffer | 1X | 1 | 420 | |
| DTT (100mM) | 8.33mM | 0.25 | 105 | |
| Betaine (5M) | 1.66M | 1 | 420 | |
| MgCl2 (1M) | 10mM | 0.03 | 12.6 | |
| TSO (100uM) | 1.66µM | 0.05 | 21 | |
| Nuclease free H2O | - | 0.495 | 207.9 | |
| Total | 3 | 1260 |
Dispense 3 µL per well . Total reaction volume will be 5µl.
Cover plate with new film and spin down.
Incubate in thermocycler
42 °C 01:30:00
70 °C 00:05:00
4 °C hold
cDNA preamplification
| A | B | C | D | |
| Reaction conc. | µl per reaction | 384w plate (420x) | ||
| Nuclease free H2O | - | 0.82 | 345 | |
| Kapa HiFi HotStart ReadyMix (2X) | 1X | 6 | 2520 | |
| IS_PCR primer (10µM) | 0.1µM | 0.12 | 50.4 | |
| Lambda Exonuclease (10u/µl) | 0.05 units | 0.06 | 25.2 | |
| Total | 7 | 2940 | ||
Dispense 7 µL per well . Total reaction volume will be 12µl.
Vortex, spin down. Cover with new lid. Incubate in thermocycler with the following program:
| A | B | C | D | |
| Step | Temperature | Time | Cycles | |
| Lambda exonuclease | 37ºC | 30 min | 1x | |
| Initial denaturation | 95ºC | 3 min | 1x | |
| Denaturation | 98ºC | 20 sec | 18-24x | |
| Annealing | 67ºC | 15 sec | ||
| Elongation | 72ºC | 4 min | ||
| Final elongation | 72ºC | 5 min | ||
| 4C | Hold |
Note
NOTE: The number of cycles of pre-amplification will be different for different cell types. We suggest running a pilot (ideally qPCR-monitored to determine inflection point, for example by using 1X dsGreen to the reaction above)
cDNA cleanup
Using 20% SPRI-bead solution (as in step10 for DNA library cleanup).
1. Add 0.7X volume of SPRI beads per well. Mix well by pipetting
2. Incubate 00:05:00 Room temperature
3. Place on magnetic stand for 00:03:00
4. Carefully remove supernatant
5. Add 40 µL 80% EtOH and incubate 00:00:30
6. Remove EtOH (without disturbing the beads)
7. Wash again with EtOH. Make sure to remove well.
8. Allow beads to air-dry for 00:05:00 -00:10:00 . Take care not to over-dry the beads.
9. Remove plate from magnetic stand
10. Elute beads in 12 µL EB or TE buffer Mix well by pipetting
11. Incubate 00:05:00 Room temperature
12. Place on magnetic plate for 00:03:00
13. Optional: Carefully remove supernatant to the elution plate. cDNA plates can also be stored at -20C with beads.
31m 30s
cDNA quantification
Option 1: Measure concentration of random wells using Qubit HS dsDNA, adapted to a 96-well plate reader.
1. Add 98.5 µL of 1X Qubit HS dsDNA solution (or mix dye and buffer separately) to a flat-bottom, black plate
2. Add 1.5 µL of cDNA sample
3. Add Standards (NOTE: We make a 8-step ladder from 0ng/µl --> 10ng/µl Qubit Standard DNA in TE buffer)
4. Read in plate reader using 485nM excitation/528nm emission
5. Calculate cDNA concentration from linear model of Standards ladder
Option 2: Measure full plate using Qubit HS dsDNA in black, flat-bottom 384-well plate
1. Add 20 µL 1X Qubit HS dsDNA solution
2. Add 1 µL cDNA sample
3-5 as above
(optional) cDNA quality control
Using Agilent HS 5000 DNA chips (or equivalent)
Example of a single immune (=small) cell cDNA profile (cytosolic fraction from DNTR protocol)
Make cDNA dilution plate
Dilute cDNA based on average concentration from Qubit measurements.
Target concentration 150 pg per µl in 15 µL (optionally in same plate)
Optional: if using a 384w-plate reader, one can normalize each well to 150pg/µl with variable water addition.
cDNA tagmentation
cDNA tagmentation
Prepare Tn5 master mix
Let TAPS-PEG equilibrate at 37ºC and mix well before use.
| A | B | C | D | |
| Reagent | Reaction conc. | µl per reaction | 384w plate (420x) | |
| Nuclease free H2O | - | 0.750 | 315 | |
| TAPS-PEG (50mM TAPS, 25mM MgCl2, 40% PEG-8000) | 10mM TAPS 5mM MgCl2 8% PEG-8000 | 0.500 | 210 | |
| psfTn5, loaded with 50µM MEDS-A/B | 0.250 | 105 | ||
| Total | 1.5 | 630 | ||
Dispense 1.5 µL per well in a new plate (tagmentation plate)
Add 1 µL cDNA (normalized to 150pg/µl)
Mix well by vortexing plate. Cover with new lid and spin down.
Incubate in thermocycler at 55 °C 00:10:00
Remove immediately and stop reaction by adding 1 µL per well of 0.1% SDS.
Vortex, spin down and incubate 00:10:00 at 55 °C
20m
cDNA library PCR and barcoding
cDNA library PCR and barcoding
Make PCR master-mix
| A | B | C | D | |
| Reagent | Reaction conc. | µl per reaction | 384w plate (420x) | |
| H2O | - | 4.85 | 2037 | |
| KAPA HiFi Buffer (5X) | 1X | 2.5 | 1050 | |
| dNTP (10mM/each) | 0.3mM/each | 0.3 | 126 | |
| KAPA enzyme (1u/µl) | 0.02u/µl | 0.2 | 84 | |
| Tween-20 (10%) | 0.12% | 0.15 | 63 | |
| Total | 8 | |||
Dispense 8 µL per well to tagmentation plate (containing 3.5µl sample after step 23)
Add primers/barcodes 1 µL per well (from 384-well index plates, with 3.75µM/each forward/reverse primers; see oligos; final primer concentration 0.3µM per primer and reaction).
Total reaction volume is 12.5µl (3.5µl sample + 9µl PCR mix and primers).
Vortex. Spin down and cover. Incubate in thermocycler as below:
| Step | Temperature | Time | Cycles | |
| Gap fill | 72ºC | 3 min | 1x | |
| First denature | 95ºC | 30 sec | 1x | |
| Denature | 95ºC | 15 sec | 12x | |
| Anneal | 67ºC | 30 sec | ||
| Extend | 72ºC | 45 sec | ||
| Final extension | 72ºC | 4 min | 1x | |
| 4-10ºC | hold |
cDNA library pooling and clean-up
cDNA library pooling and clean-up
Pool 2.5 µL from each well to an 1.5ml Eppendorf tube
Library cleanup (as for DNA libraries)
We prepare SPRI-beads in 20% PEG-8000 solution as in: https://openwetware.org/wiki/SPRI_bead_mix#Ingredients_for_50_mL_2
1. Add 0.9X SPRI-beads in 20% PEG solution. Incubate for 00:05:00 Room temperature
2. Place on magnetic rack 00:03:00
3. Remove supernatant
4. Add 1 volume 80% EtOH (fresh). Incubate for 00:00:30
5. Remove supernatant
6. Repeat EtOH wash
7. Air dry for 00:10:00 - - 00:15:00
8. Re-suspend beads thoroughly in 100 µL EB or TE buffer
9. Repeat cleanup (from step 1-7) and elute in 30 µL EB or TE buffer
Pooled library QC
Pooled cDNA library of 784 cells on HS D5000 Agilent tapestation
Citations
Step 7
Picelli S, Björklund AK, Reinius B, Sagasser S, Winberg G, Sandberg R. Tn5 transposase and tagmentation procedures for massively scaled sequencing projects.
https://doi.org/10.1101/gr.177881.114