TITLE: single-cell Micro-C protocol
AUTHORS: Honggui Wu, Longzhi Tan

[DESCRIPTION]
Here we present a single-cell 3D genome mapping assay with improved spatial resolution with the help of micrococcal nuclease (MNase), termed single-cell Micro-C (scMicro-C). To achieve scMicro-C, we made three improvements. First, we titrated MNase digestion to reduce DNA loss and produce proper DNA fragments. Second, we solubilized chromatin with an ionic detergent, sodium dodecyl sulfate (SDS), which dramatically improved ligation efficiency. Third, we adopted our high-coverage transposon-based whole-genome amplification method, META (19), using Tn5 (20), to enhance the detection of chromatin “contacts.” scMicro-C retains the high signal-to-noise and high-resolution characteristics of bulk Micro-C, as indicated the accurate detection chromatin loops and other fine-scale chromatin structures. Furthermore, scMicro-C retains nucleosome and other chromatin-bound proteins (e.g., transcription factor) occupancy information. With our previously developed Dip-C algorithm,  we confirmed that scMicro-C enables the reproducible reconstruction of 3D genome structures in single-cell at 5 kb resolution. With such kilobase 3D structures, we were able to explore the fine-scale chromatin folding in individual cells.

[STEPS]
SECTION: Prepare Micro-C Buffers
9. Prepare Micro-C Buffers
SECTION: Prepare Micro-C Buffers
9.1. 10 mL Prepare Micro-C Buffer 1 (300 µL)
50 µL 5 M NaCl (Invitrogen AM9760G; final 50 mM)
50 µL 1 M Tris pH 7.5 (Invitrogen 15567027; final 10 mM)
25 µL 1 M MgCl2 (Invitrogen AM9530G; final 5 mM)
5 µL 1 M CaCl2 (Sigma 21115; final 1 mM)
50 µL 5% Digitonin (Sigma D141-100MG, final: 0.05%)
50 µL 100 x protease inhibitor (Roche 4693159001) (final: 1 X)
4.77 mL nuclease-free H2O








SECTION: Prepare Micro-C Buffers
9.2. Prepare Micro-C Buffer 2 (10 mL)
100 µL 5 M NaCl (Invitrogen AM9760G; final 50 mM) 
100 µL 1 M Tris pH 7.5 (Invitrogen 15567027; final 10 mM)
100 µL 1 M MgCl2 (Invitrogen AM9530G; final 10 mM)
9.7 mLnuclease-free H2O


  




SECTION: Prepare Micro-C Buffers
9.3. Prepare Micro-C Buffer 3 (10 mL)
500 µL 1 M Tris pH 7.5 (Invitrogen 15567027; final 50 mM)
100 µL 1 M MgCl2 (Invitrogen AM9530G; final 10 mM)
9.4 mL nuclease-free H2O



SECTION: MNase Digestion
12. Resuspend in 100 µL Micro-C Buffer 1, incubate on ice for 20 min, and aspiration every 5 min.

SECTION: Crosslink Cells for Micro-C
1. Prepare 1 x BSA in PBS for Washing.
Add 50 µL 10% BSA (MACS 130-091-376, final 1x) to 50 mL 1x PBS.

SECTION: Crosslink Cells for Micro-C
2. Prepare 1% Paraformaldehyde (1 mL for 1 million cells), recover to room temperature.
1 mL 32% PFA (EMS 15714, final 1%)
31 mL 1x PBS


SECTION: Crosslink Cells for Micro-C
4. Harvest cells.
Collect cell growth with appropriate density and high viability.
Wash cells once with 1x PBS.
SECTION: Crosslink Cells for Micro-C
5. Fixation.
Resuspend cell pellets in 1% PFA, and incubate at room temperature with rotation for 10 min.
 At the end of incubation, add 2 M Tris-HCl ph 7.5 to a final concentration of 0.75 M, and incubate at RT for 5 min.
Centrifuge at 3000 x g for 5 min in a swing bucket centrifuge.
SECTION: Crosslink Cells for Micro-C
6. Remove the supernatant, then wash twice with 5 mL ice-cold 1 x BSA in PBS.

SECTION: Crosslink Cells for Micro-C
7. Crosslink with DSG.
Resuspend in 3 mM DSG, and incubate at RT for 45 min with rotation.
At the end of incubation, add 2 M Tris-HCl ph 7.5 to a final concentration of 0.75 M, and incubate at RT for 5 min.
Centrifuge at 3000 x g for 5 min in a swing bucket centrifuge.


SECTION: Crosslink Cells for Micro-C
3. Prepare 3 mM DSG (1 mL for 1 million cells).
Dissolve 50 mg DSG (Thermo Fisher 20593) with 511 µL DMSO to a final concentration of 300 mM.
Add 300 µL 300 mM DSG to 29.7 mL 1 x PBS to a final concentration of 3 mM, protected from light. 

SECTION: Crosslink Cells for Micro-C
8. Count cells and aliquot.
Remove the supernatant, then wash twice with 5 mL ice-cold 1 x BSA in PBS.
Count cell number, then aliquot 1 million cells each to 1.5 mL tube.
Store at -80°C (up to a year).

SECTION: MNase Digestion
13. Centrifuge at 800 x g at 4°C for 5 min.
SECTION: MNase Digestion
10. Dilute MNase.
Make MNase Dilution Buffer (10 mM Tris-HCl ph 7.5, 50 mM NaCl, 1 mM EDTA, 50% Glycerol).
Aliquot 10,000 Unit MNase (NEB M0247S, 2,000 U/µL) to 500 µLMNase Dilution Buffer to a final concentration of 20 U/µL.
Store at -80°C for several month.

SECTION: MNase Digestion
11. Thaw 5 tubes of cell pellets on ice.
SECTION: MNase Digestion
15. Check Digestion Efficiency
Take 5 µL above sample to check digestion efficiency.
Add 85 ul 1 x PBS, 5 ul 10% SDS (Sigma, 71736-100ML), and 5 ul 20 mg/mL ProteaseK (QIAGEN, 19131).
incubate at 65°C for 2 hr.
Purify with DNA clean column (ZYMO, D4014).
Run capillary electrophoresis (Fragment Analyzer/TepeStation/Bioanalyzer) to analyze digestion efficiency.
 

SECTION: MNase Digestion
14. Take 5 ul to check digestion efficiency, remaining centrifuge at 800 x g at 4°C for 5 min. Carefully discard the supernatant, and wash once with 500 µLMicro-C Buffer 2. Store at -80°C (stable for up to several weeks) or proceed to End Repair.

SECTION: End Repair
16. Thaw the cell pellet (with the most appropriate fragment size) on ice.
SECTION: End Repair
17. Prepare 0.5% SDS.
Add 25 µL 10% SDS to 475 µL Micro-C Buffer 2 to a final concentration of 0.5%.

SECTION: End Repair
18. Solubilize Chromatin.
Resuspend the cell pellets in 50 µL 0.5% SDS.
Incubate at 62°C for 10 min.
At the end of incubation, add 50 µL 5% Triton X-100 to quench SDS. Incubate at 37°C for 15 min.

SECTION: End Repair
19. Centrifuge at 800 x g for 5 min, carefully remove supernatant.
SECTION: End Repair
20. Prepare End Repair Mix 1 (45 µL each sample).
5 µL 10 X NEBuffer 2.1 (NEB B7202S)
1 µL100 mM ATP (Thermo R0441)
2.5 µL 100 mM DTT (final: ~5 mM)
2.5 µL 10 U/uL T4 PNK (NEB M0201S) (final: ~0.5 U/uL)
34 µL nuclease-free H2O





SECTION: End Repair
21. Resuspend in 45 µL End Repair Mix1, and incubate at 37°C for 15 min. 

SECTION: End Repair
22. At the end of incubation, add 5 µL 5 U/uL Klenow Fragment (NEB M0210S), incubate at 37°C for another 15 min.

SECTION: End Repair
23. Prepare End Repair Mix2 (25 µLeach sample)
2.5 µL 10 X T4 DNA Ligase Buffer (NEB B0202S)
0.5 µL 10 mM (each) dNTP (NEB N0447S)
0.125 µL 20 mg/mL BSA (NEB B9000S)
21.875 µL nuclease-free H2O




SECTION: End Repair
24. After Klenow incubation, add 25 µL End Repair Mix2 to each tube, incubate at 23°C for 45 min (shake 30 s and pause 2 min). 

SECTION: End Repair
25. Add 5 ul 0.5 M EDTA (Invitrogen AM9260G).
SECTION: End Repair
26. Incubate at 65°C for 20 min.
SECTION: Proximity Ligation
28. Prepare Ligation Mix (250 µL each sample).
25 µL 10 X T4 DNA Ligase Buffer (NEB B0202S)
1.25 µL 20 mg/mL BSA (NEB B9000S) 
12.5 µL 400 U/uL T4 DNA Ligase (NEB M0202S)
211.25 µL nuclease-free H2O



SECTION: End Repair
27. Wash once with 1 mL Micro-C Buffer 3.

SECTION: Proximity Ligation
29. Resuspend the cell pellet in 250 µL Ligation Mix.

SECTION: Proximity Ligation
30. Take 25 µL as ligation efficiency control.

SECTION: Proximity Ligation
31. Check Ligation Efficiency
Take 25 µL ligation product above sample to check ligation efficiency.
Add 65 ul 1 x PBS, 5 ul 10% SDS (Sigma, 71736-100ML), and 5 ul 20 mg/mL ProteaseK (QIAGEN, 19131).
incubate at 65°C for 2 hr.
Purify with DNA clean column (ZYMO, D4014).
Run capillary electrophoresis (Fragment Analyzer/TepeStation/Bioanalyzer) to analyze ligation efficiency.
   

SECTION: Single-cell Amplification
34. Pick single cells.
Use mouth pipette or Fluorescence-activated cell sorting (FACS) to pick single cell into single PCR tubes or 96-well plates containing 2 µL cell lysis buffer.

SECTION: Single-cell Amplification
35. Cell lysis.
Incubate as:
    50℃, 1 hr
    65℃, 1 hr
    70℃, 15 min
Stored at -80℃ or proceed to transposon-based amplification.
SECTION: Single-cell Amplification
36. Transposition (8 µLfor each well, below recipe for 1 96-well plate).
Prepare Transposition Mix
440 µL 2 X Transposition Buffer
Add META transposome* to a final concentration of 0.3 nM
Add nuclease-free H2O to 880 µL
* Transposome: META transposome is optional. Alternatively, Nextera transposome can be used (if using Nextera procedure, please refer to "Dip-C (Part 2: Whole-genome Amplification with Nextera)") on protocol.io.
2. Add 8 µL Transposition Mix to each well with a multi-channel pipette, vortex to mix.
3. Incubate at 55℃ for 10 min.



SECTION: Single-cell Amplification
37. Tn5 Stop.
Prepare Tn5 Removal Mix (250 mM NaCl, 37.5 mM EDTA, 2 mg/mL QIAGEN protease).
Add 2 µL Tn5 Removal Mix to each tube, and mix thoroughly.
Incubate as:
      50℃, 30 min
      70℃, 15 min

SECTION: Single-cell Amplification
33. Prepare Reagents.
SECTION: Single-cell Amplification
33.1. Prepare 60 mg/mL Protease.
Dissolve 1 vial QIAGEN protease (QIAGEN 19155) in 2.78 mL 50% glycerol.
Aliquot and store at -80°C (stable for up half a year).
SECTION: Single-cell Amplification
33.2. Cell Triton Lysis Buffer (1 mL).
10 µL 1 M Tris pH 8.0 (Invitrogen AM9855G, final 10 mM)
4 µL 5 M NaCl (Invitrogen AM9760G, final 20 mM)
2 µL 0.5 M EDTA (Invitrogen AM9260G, final 1 mM)
10 µL 10% Triton X-100 (Sigma 93443, final 0.1%)
25 µL 1 M DTT (Sigma 646563, final 25 mM)
5 µL 100 µM Carrier ssDNA (final 100 nM)
Add H2O to 1 mL
25 µL 60 mg/mL QIAGEN protease (QIAGEN 19155, final 1.5 mg/mL) (freshly add when used)

 



SECTION: Single-cell Amplification
33.3. 2 X Transposition Buffer (1 mL)
20 µL 1 M Tris-HCl pH 8.0 (Invitrogen AM9855G, final 20 mM)
10 µL 1 M MgCl2 (Thermo Fisher AM9530G, final 10 mM)
400 ul 40% (w/w) Polyethylene glycol 8000 (Sigma P1458, final 16% (w/w) )
570 µLnuclease-free H2O
Store at -20℃ if needed



SECTION: Single-cell Amplification
38. Pre-amplification.




SECTION: Single-cell Amplification
39. Cell Barcoding PCR.
Add 1 µL individual 50 μM barcoded META16-ADP1 Primer and 1 µL individual 50 μM indexed META16-ADP2 Primer Mix to each well with a multi-channel pipette.
Incubate as:
     98℃, 30 s
     3 cycles of [98℃ for 10 s, 62℃ for 30 s, 72℃, 2 min]
     65℃, 5 min
  

SECTION: Single-cell Amplification
40. Purification.
Pool a 96-well plate to purify.
Purify with clean column (ZYMO, D4014).
Quantify with Qubit Fluorometer.
SECTION: Single-cell Amplification
41. Library preparation.
Take 400 ng amplified product for each plate as input.
Prepare Library Preparation Mix (50 µL2 X Q5 High-fidelity Mater Mix, template (400 ng), 5 µL 10 µM Indexed i5 primer, 5 µL 10 µM Indexed i7 primer (Vazyeme N321/N322), nuclease-free H2O to 100 µL)
Amplified as:
      98℃, 30 s
      2 cycles of [98℃, 10 s, 68℃, 30 s, 72℃, 2 min] 
      72℃, 5 min
Purify with PCR clean column (ZYMO D4014), then purify with 0.75 x AMPUre XP beads to select > 300 bp fragments for sequencing.




SECTION: Single-cell Amplification
32. Primers
META 16 sequence

 
META Transposon
 

 First Step PCR Primers

 
Here we use 12 x 8 barcode combinations to distinguish individual cells.
META16-ADP1 cell barcode
 
META16-ADP2 cell barcode

 
Second Step Barcoded PCR Primers

 

SECTION: Single-cell Amplification
38.1. Prepare Preamplification Mix (16 µL each well, below recipe for 1 96-well plate)
1540 µL 2 X Q5 High-fidelity Master Mix (NEB M0492L)
98.6 µL 50 µM First Step PCR Primers (final 0.1 µM each)
55 µL 100 mM MgCl2
66.4 µL nuclease-free H2O
SECTION: Single-cell Amplification
38.2. Add 16 µL Preamplification Mix each well with a multi-channel pipette, and vortex to mix.

SECTION: Single-cell Amplification
38.3. Amplified as:
    72℃, 5 min
    98℃, 30 s
    12 cycles of [98℃ for 10 s, 62℃ for 30 s, 72℃, 2 min]
    65℃, 5 min.
SECTION: Single-cell Amplification
42. Sequencing.
    META library only has 20% valid fragments having both P7 and P5 on either side, need to RUN qPCR quantify before loading.