TITLE: Direct nuclear tagmentation and RNA-sequencing (DNTR-seq)
AUTHORS: Vasilios Zachariadis, Huaitao Cheng, Nathanael Andrews, Martin Enge

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Prepare lysis buffer plates for cell sorting]
Prepare lysis buffer mixNOTE: Reagents are prepared on ice, working quickly. ERCC is stored in single-use aliquots at  , thawed on ice and added last.   ABCD1ReagentReaction conc.µL per reaction384w plate2Nuclease free H2O-1.9657863RNase Inhibitor (40u/µL)1 unit/µL0.075304ERCC (1:1 200 000)-0.075305Triton-X100 (10% solution)0.2%0.06246dNTP (10mM each)2.5mM/each0.753007Oligo-dT (100µM)2.5µM0.0753089To dispense31200Add to each well. Cover with appropriate lids. Spin down.Snap freeze on dry ice. Store until use at
-80 °C
ABCD1ReagentReaction conc.µL per reaction384w plate2Nuclease free H2O-1.9657863RNase Inhibitor (40u/µL)1 unit/µL0.075304ERCC (1:1 200 000)-0.075305Triton-X100 (10% solution)0.2%0.06246dNTP (10mM each)2.5mM/each0.753007Oligo-dT (100µM)2.5µM0.0753089To dispense31200
[lysis buffer mix]
-80 °C
?. [Sort single-cells]
Sort single cells into Immediately seal with appropriate seals (approved for -80C > 100C) and centrifuge at Snap freeze on dry ice. Store until use at
[lysis buffer mix]
Centrifuge: 2000 34, 5 min, 4 10
-80 °C
?. [Separation of nuclear and cytosolic fractions]
Thaw plate on ice. Centrifuge at . Keep on ice.
Centrifuge: 500 34, 5 min, 4 10
?. [Separation of nuclear and cytosolic fractions]
Transfer   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.
2 µl
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.
?. [Separation of nuclear and cytosolic fractions]
Spin down and freeze nuclear fraction at  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
-20 °C
-80 °C
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]
Using plate with nuclear fraction, with remaining volume 1µl/well. Proteinase K treatment1. Dilute Proteinase K (stock 20mg/ml) to 0.2mg/ml by 30mM Tris-HCl pH8.02. Add  diluted Proteinase K (0.2mg/ml) to each well. Makes 0.13mg/ml reaction concentration.3. Incubate in thermocycler at: - - -
2 µl
50 °C
80 °C
[hold]
?. [Single-cell genomic libraries]
Tn5 digestionTn5 is produced from psfTn5 (Addgene #79107), purified to ~3mg/ml and assembled with Illumina Tn5 adapters (see oligos) as in Picelli et al, 2014.
?. [Single-cell genomic libraries]
Prepare 2X Tn5 Buffer. Keep assembled Tn5 enzyme (Picelli et al, 2014) on ice block and add last.  ABCD1ReagentReaction concµL per reaction384w plate25X TAPS-PEG
(50mM TAPS,
25mM MgCl2,
40% PEG-8000)10mM TAPS
5mM MgCl2
8% PEG-80001.66723psfTn5, loaded with 50µM MEDS-A/B0.1424Nuclease free H2O3.3138656To dispense52100Add  per well. Vortex and spin down plate.
ABCD1ReagentReaction concµL per reaction384w plate25X TAPS-PEG
(50mM TAPS,
25mM MgCl2,
40% PEG-8000)10mM TAPS
5mM MgCl2
8% PEG-80001.66723psfTn5, loaded with 50µM MEDS-A/B0.1424Nuclease free H2O3.3138656To dispense52100
5 µl
NOTE: Buffer contains PEG, which is viscous. 5X TAPS-PEG buffer should be allowed to assume room temperature before dispensing to allow proper mixing.
?. [Single-cell genomic libraries]
Incubate in thermocycler: Remove immediately and stop reaction by adding  of 0.2% SDS. Vortex, spin down and incubate  at
55 °C
[per well]
55 °C
?. [Single-cell genomic libraries]
PCR amplification and barcoding1. Prepare PCR master-mix  ABCD1ReagentReaction conc.µl per reaction384w plate2Nuclease free H2O-3.212803KAPA HiFi Buffer (5X)1X416004dNTP (10mM/each)0.3mM/each0.62405KAPA enzyme (1u/µl)0.02u/µl0.41606Tween-20 (10%)0.1%0.28078To dispense8.433602. Dispense 3. Add primers/barcodes  (from 384-well index plates, with 3.75µM/each forward/reverse primers; see oligos). Total reaction volume is now 20µl (10µl sample + 10µl PCR mix and primers). 4. Vortex plate, spin down and incubate in thermocycler with the following program:  ABCD1StepTemperatureTimeCycles2Gap fill72ºC3 min1x3First denature95ºC30 sec1x4Denature95ºC15 sec18x5Anneal67ºC30 sec6Extend72ºC45 sec7Final extension72ºC4 min1x84-10ºChold
ABCD1ReagentReaction conc.µl per reaction384w plate2Nuclease free H2O-3.212803KAPA HiFi Buffer (5X)1X416004dNTP (10mM/each)0.3mM/each0.62405KAPA enzyme (1u/µl)0.02u/µl0.41606Tween-20 (10%)0.1%0.28078To dispense8.43360
[per well]
[per well]
ABCD1StepTemperatureTimeCycles2Gap fill72ºC3 min1x3First denature95ºC30 sec1x4Denature95ºC15 sec18x5Anneal67ºC30 sec6Extend72ºC45 sec7Final extension72ºC4 min1x84-10ºChold
?. [Single-cell genomic libraries]
Pool  into a 1.5mL Eppendorf tube.
[from each well]
?. [Single-cell genomic libraries]
Library cleanupWe prepare SPRI-beads in 20% PEG-8000 solution as in: https://openwetware.org/wiki/SPRI_bead_mix#Ingredients_for_50_mL_21. Add 0.9X SPRI-beads in 20% PEG solution. Incubate for 2. Place on magnetic rack 3. Remove supernatant4. Add 1 volume 80% EtOH (fresh). Incubate for 5. Remove supernatant6. Repeat EtOH wash7. Air dry for - 8. Re-suspend beads thoroughly in 9. Repeat cleanup (from step 1-7) and elute in
0 Room temperature
[EB or TE buffer]
[EB or TE buffer]
?. [Single-cell genomic libraries]
(optional) Quality control of DNA librariesUsing Agilent HS 5000 DNA chips (or equivalent)This library was sequenced on a NextSeq loading 2.5pM based on a peak of 334bp. Sequencing was paired-end 37bp, 8bp dual index.
?. [Reverse transcription and cDNA amplification]
Following step 4, cytosolic/RNA fraction plate contains 2µl solution per well. Primer annealingThaw plate. Spin down. Incubate in thermocycler at  for . Remove to ice immediately.
72 °C
?. [Reverse transcription and cDNA amplification]
Prepare RT master-mix  ABCD1ReagentReaction conc.µl per reaction384w plate23SMARTScribe RT (100u/µl)15u/µl0.475199.54RNase Inhibitor (40u/µl)1.66u/µl0.12552.555X First Strand buffer1X14206DTT (100mM)8.33mM0.251057Betaine (5M)1.66M14208MgCl2 (1M)10mM0.0312.69TSO (100uM)1.66µM0.052110Nuclease free H2O-0.0729.41112Total31260Dispense . Total reaction volume will be 5µl. Cover plate with new film and spin down.
ABCD1ReagentReaction conc.µl per reaction384w plate23SMARTScribe RT (100u/µl)15u/µl0.475199.54RNase Inhibitor (40u/µl)1.66u/µl0.12552.555X First Strand buffer1X14206DTT (100mM)8.33mM0.251057Betaine (5M)1.66M14208MgCl2 (1M)10mM0.0312.69TSO (100uM)1.66µM0.052110Nuclease free H2O-0.0729.41112Total31260
[per well]
?. [Reverse transcription and cDNA amplification]
Incubate in thermocycler
42 °C
70 °C
[hold]
?. [Reverse transcription and cDNA amplification]
cDNA preamplification   ABCD1Reaction conc.µl per reaction384w plate2Nuclease free H2O-1.0688470.253Kapa HiFi HotStart ReadyMix (2X)1X6.2527504IS_PCR primer (10µM)0.1µM0.125555Lambda Exonuclease (10u/µl)0,045u/µl0.0562524.7567Total7.50003300 Dispense  . Total reaction volume will be 12.5µl.
ABCD1Reaction conc.µl per reaction384w plate2Nuclease free H2O-1.0688470.253Kapa HiFi HotStart ReadyMix (2X)1X6.2527504IS_PCR primer (10µM)0.1µM0.125555Lambda Exonuclease (10u/µl)0,045u/µl0.0562524.7567Total7.50003300
[per well]
?. [Reverse transcription and cDNA amplification]
Spin down. Cover with new lid. Incubate in thermocycler with the following program:   ABCD1StepTemperatureTimeCycles2Lambda exonuclease37ºC30 min1x3Initial denaturation95ºC3 min1x4Denaturation98ºC20 sec18-24x5Annealing67ºC15 sec6Elongation72ºC4 min7Final elongation72ºC5 min84CHold
ABCD1StepTemperatureTimeCycles2Lambda exonuclease37ºC30 min1x3Initial denaturation95ºC3 min1x4Denaturation98ºC20 sec18-24x5Annealing67ºC15 sec6Elongation72ºC4 min7Final elongation72ºC5 min84CHold
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)
?. [Reverse transcription and cDNA amplification]
cDNA cleanupUsing 20% SPRI-bead solution (as in step10 for DNA library cleanup). 1. Add 0.7X volume of SPRI beads per well. Mix well by pipetting2. Incubate 3. Place on magnetic stand for 4. Carefully remove supernatant5. Add  80% EtOH and incubate 6. Remove EtOH (without disturbing the beads)7. Wash again with EtOH. Make sure to remove well.8. Allow beads to air-dry for  -9. Remove plate from magnetic stand10. Elute beads in  Mix well by pipetting11. Incubate 12. Place on magnetic plate for 13. Optional: Carefully remove supernatant to the elution plate
0 Room temperature
40 µl
[EB or TE buffer]
0 Room temperature
?. [Reverse transcription and cDNA amplification]
cDNA quantificationWe measure concentration of random wells using Qubit HS dsDNA, adapted to a 96-well plate reader. 1. Add  of 1X Qubit HS dsDNA solution (or mix dye and buffer separately) to a flat-bottom, black plate2. Add  of cDNA sample3. Add Standards (NOTE: We make a 8-step ladder from 0ng/µl --> 10ng/µl Qubit Standard DNA in TE buffer)3. Read in plate reader using 485nM excitation/528nm emission 4. Calculate cDNA concentration
98.5 µl
1.5 µl
?. [Reverse transcription and cDNA amplification]
(optional) cDNA quality controlUsing Agilent HS 5000 DNA chips (or equivalent)
?. [Reverse transcription and cDNA amplification]
Make cDNA dilution plateDilute cDNA based on average concentration from Qubit measurements. Target concentration  in   (optionally in same plate)
[per µl]
15 µl
?. [cDNA tagmentation]
Prepare Tn5 master mixLet TAPS-PEG equilibrate at 37ºC and mix well before use.  ABCD1ReagentReaction conc.µl per reaction384w plate2Nuclease free H2O-1.0505253TAPS-PEG
(50mM TAPS,
25mM MgCl2,
40% PEG-8000)10mM TAPS
5mM MgCl2
8% PEG-80000.5002504psfTn5, loaded with 50µM MEDS-A/B0.25012556Total1.800900Dispense  in a new plate (tagmentation plate)
ABCD1ReagentReaction conc.µl per reaction384w plate2Nuclease free H2O-1.0505253TAPS-PEG
(50mM TAPS,
25mM MgCl2,
40% PEG-8000)10mM TAPS
5mM MgCl2
8% PEG-80000.5002504psfTn5, loaded with 50µM MEDS-A/B0.25012556Total1.800900
[per well]
?. [cDNA tagmentation]
Add  cDNA (normalized to 150pg/µl)Mix well by vortexing plate. Cover with new lid and spin down.
0.7 µl
?. [cDNA tagmentation]
Incubate in thermocycler at Remove immediately and stop reaction by adding  of 0.2% SDS. Vortex, spin down and incubate  at
55 °C
[per well]
55 °C
?. [cDNA library PCR and barcoding]
Make PCR master-mix  ABCD1ReagentReaction conc.µl per reaction384w plate (420x)2H2O-13.2555653KAPA HiFi Buffer (5X)1X521004dNTP (10mM/each)0.3mM/each0.753155KAPA enzyme (1u/µl)0.02u/µl0.521067Total19.5 Dispense  to tagmentation plate (containing 3.5µl sample after step 23)
ABCD1ReagentReaction conc.µl per reaction384w plate (420x)2H2O-13.2555653KAPA HiFi Buffer (5X)1X521004dNTP (10mM/each)0.3mM/each0.753155KAPA enzyme (1u/µl)0.02u/µl0.521067Total19.5
[per well]
?. [cDNA library PCR and barcoding]
Add primers/barcodes  (from 384-well index plates, with 3.75µM/each forward/reverse primers; see oligos). Total reaction volume is 25µl (3.5µl sample + 21.5µl PCR mix and primers).
[per well]
?. [cDNA library PCR and barcoding]
Vortex. Spin down and cover. Incubate in thermocycler as below:   ABCD1StepTemperatureTimeCycles2Gap fill72ºC3 min1x3First denature95ºC30 sec1x4Denature95ºC15 sec12x5Anneal67ºC30 sec6Extend72ºC45 sec7Final extension72ºC4 min1x84-10ºChold
ABCD1StepTemperatureTimeCycles2Gap fill72ºC3 min1x3First denature95ºC30 sec1x4Denature95ºC15 sec12x5Anneal67ºC30 sec6Extend72ºC45 sec7Final extension72ºC4 min1x84-10ºChold
?. [cDNA library pooling and clean-up]
Pool  from each well to an 1.5ml Eppendorf tube
2.5 µl
?. [cDNA library pooling and clean-up]
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_21. Add 0.9X SPRI-beads in 20% PEG solution. Incubate for 2. Place on magnetic rack 3. Remove supernatant4. Add 1 volume 80% EtOH (fresh). Incubate for 5. Remove supernatant6. Repeat EtOH wash7. Air dry for - 8. Re-suspend beads thoroughly in 9. Repeat cleanup (from step 1-7) and elute in
0 Room temperature
[EB or TE buffer]
[EB or TE buffer]
?. [cDNA library pooling and clean-up]
Pooled library QC