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

[DESCRIPTION]
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.

[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. 

[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. 

[STEPS]
SECTION: Prepare lysis buffer plates for cell sorting
1. 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. 
 
       rxn13115228848006000H2O1,313,931509,12377,2862887860RNase Inhibitor0,050,1557,614,4240300ERCC (1:1 200 000)0,050,1557,614,424030010% Triton0,040,1246,0811,5219224010mM dNTP0,51,557614424003000100uM 5'Biot-dT0,050,1557,614,4240300 Total262304576960012000
 
Add 3 µLto each well. Cover with appropriate lids. Spin down.
Snap freeze on dry ice. Store until use at -80 °C 
SECTION: Sort single-cells
2. Sort single cells into 3 µL
Immediately seal with appropriate seals (approved for -80C > 100C) and centrifuge at 2000 x g, 5 min, 4 °C 
Snap freeze on dry ice. Store until use at -80 °C 
SECTION: Separation of nuclear and cytosolic fractions
3. Thaw plate on ice. 
Centrifuge at 500 x g, 5 min, 4 °C. 
Keep on ice. 
SECTION: Separation of nuclear and cytosolic fractions
4. 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.
 

SECTION: Separation of nuclear and cytosolic fractions
5. 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 
 


SECTION: Single-cell genomic libraries
6. 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 60 min 
- 80 °C 30 min 
- 4 °C
SECTION: Single-cell genomic libraries
7. 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. 
 


SECTION: Single-cell genomic libraries
7.1. Prepare 2X Tn5 Buffer. Keep assembled Tn5 enzyme (Picelli et al, 2014) on ice block and add last.
 
      12204205X TAPS-PEG 1.6352672Buffer is 50mM TAPS, 25mM MgCl2, 40% PEG 8k. Final concentration is 8% PEG, 5mM MgCl2, 10mM TAPS.psfTn5, loaded with 50µM MEDS-A/B0.12242Conc equivalent to 0.1µl in 8µl reaction. Keep in iceblock, add lastH2O3.37261386Volume511002100

Add 5 µL per well. Vortex and spin down plate. 
 

SECTION: Single-cell genomic libraries
7.2. Incubate in thermocycler: 55 °C10 min 
Remove immediately and stop reaction by adding 2 µL of 0.2% SDS. 
Vortex, spin down and incubate 10 min at 55 °C 

SECTION: Single-cell genomic libraries
8. PCR amplification and barcoding

1. Prepare PCR master-mix
   1220H2O3.27045x buffer4880dNTP0.6132KAPA0.488Tween-20, 10%0.244To dispense8.41848

2. Dispense 8 µL 

3. Add primers/barcodes 1.5 µL (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:
 
  72C, 3 min95C, 30s18 cycles of95C, 15s67C, 30s72C, 45s72C 4 min4C, hold

SECTION: Single-cell genomic libraries
9. Pool 3 µL into a 1.5mL Eppendorf tube.

SECTION: Single-cell genomic libraries
10. 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 5 min Room temperature 
2. Place on magnetic rack 3 min 
3. Remove supernatant
4. Add 1 volume 80% EtOH (fresh). Incubate for 30 s 
5. Remove supernatant
6. Repeat EtOH wash
7. Air dry for 10 min- 15 min   
8. Re-suspend beads thoroughly in 100 µL
9. Repeat cleanup (from step 1-7) and elute in 30 µL 
SECTION: Single-cell genomic libraries
11. (optional) Quality control of DNA libraries

Using Agilent HS 5000 DNA chips (or equivalent)

  
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. 

SECTION: Reverse transcription and cDNA amplification
12. Following step 4, cytosolic/RNA fraction plate contains 2µl solution per well. 

Primer annealing
Thaw plate. Spin down. Incubate in thermocycler at 72 °C for3 min . Remove to ice immediately. 

SECTION: Reverse transcription and cDNA amplification
13. Prepare RT master-mix

   1840Cell Lysis Volume2SmartScribe0.475399RNase Inhibitor0.1251055x First Strand buffer1840DTT (100mM)0.25210Betaine (5M) [fridge]1840MgCl2 (1M) [bench]0.0325.2TSO (100uM)0.0542H200.0758.8Total32520
Dispense 3 µL. Total reaction volume will be 5µl. 
Cover plate with new film and spin down.
SECTION: Reverse transcription and cDNA amplification
14. Incubate in thermocycler
42 °C90 min 
70 °C 5 min 
4 °C 
SECTION: Reverse transcription and cDNA amplification
15. cDNA preamplification 
 
   Reactions1420RT reaction52100H2O1.0688448.875Kapa HiFi HotStart ReadyMix (2x)6.252625IS_PCR primer (10uM)0.12552.5Lambda Exonuclease0.0562523.625Total7.50003150
 Dispense 7 µL . Total reaction volume will be 12µl. 

SECTION: Reverse transcription and cDNA amplification
16. Vortex, spin down. Cover with new lid. Incubate in thermocycler with the following program: 
 
  37C for 30min;95C for 3min;21 cycles of:98C for 20s67C for 15s72C for 4min72C for 5min4C hold

 

SECTION: Reverse transcription and cDNA amplification
17. 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 5 min Room temperature 
3. Place on magnetic stand for 3 min 
4. Carefully remove supernatant
5. Add 40 µL 80% EtOH and incubate 30 s 
6. Remove EtOH (without disturbing the beads)
7. Wash again with EtOH. Make sure to remove well.
8. Allow beads to air-dry for 5 min -10 min . Take care not to over-dry the beads.
9. Remove plate from magnetic stand
10. Elute beads in 12 µL Mix well by pipetting
11. Incubate 5 min Room temperature 
12. Place on magnetic plate for 3 min 
13. Optional: Carefully remove supernatant to the elution plate. cDNA plates can also be stored at -20C with beads.

SECTION: Reverse transcription and cDNA amplification
18. 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 
2. Add 1 µL 
3-5 as above
SECTION: Reverse transcription and cDNA amplification
19. (optional) cDNA quality control

Using Agilent HS 5000 DNA chips (or equivalent)
  

SECTION: Reverse transcription and cDNA amplification
20. Make cDNA dilution plate

Dilute cDNA based on average concentration from Qubit measurements.
Target concentration 150 pg 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. 

SECTION: cDNA tagmentation
21. Prepare Tn5 master mix

Let TAPS-PEG equilibrate at 37ºC and mix well before use.
 
   1500H2O1.050525TAPS-PEG0.500250tn5 stock0.250125 Total1.800900

Dispense 1.5 µL in a new plate (tagmentation plate)
SECTION: cDNA tagmentation
22. Add 1 µL cDNA (normalized to 150pg/µl)
Mix well by vortexing plate. Cover with new lid and spin down. 
SECTION: cDNA tagmentation
23. Incubate in thermocycler at 55 °C 10 min 
Remove immediately and stop reaction by adding 1 µL of 0.1% SDS. 
Vortex, spin down and incubate 10 min at 55 °C 
SECTION: cDNA library PCR and barcoding
24. Make PCR master-mix
 
   1420H2O13.2555655x buffer52100dNTPs0.75315KAPA0.5210Dispense19.50
 Dispense 8 µL to tagmentation plate (containing 3.5µl sample after step 23)

SECTION: cDNA library PCR and barcoding
25. Add primers/barcodes 1 µL (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). 
SECTION: cDNA library PCR and barcoding
26. Vortex. Spin down and cover. Incubate in thermocycler as below: 
 
  72C, 3 min95C, 30s18 cycles of95C, 15s67C, 30s72C, 45s72C 4 min4C, hold

SECTION: cDNA library pooling and clean-up
27. Pool 2.5 µL from each well to an 1.5ml Eppendorf tube

SECTION: cDNA library pooling and clean-up
28. 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 5 min Room temperature 
2. Place on magnetic rack 3 min 
3. Remove supernatant
4. Add 1 volume 80% EtOH (fresh). Incubate for 30 s 
5. Remove supernatant
6. Repeat EtOH wash
7. Air dry for 10 min- 15 min   
8. Re-suspend beads thoroughly in 100 µL
9. Repeat cleanup (from step 1-7) and elute in 30 µL 
SECTION: cDNA library pooling and clean-up
29. Pooled library QC