TITLE: MUSIC Protocol
AUTHORS: Wenxin Zhao, Zhifei Luo, Sheng Zhong

[DESCRIPTION]
Here we introduce the Multi-Nucleic Acid Interaction Mapping in Single Cell (MUSIC) technique for concurrent profiling of multiplex chromatin interactions, gene expression, and RNA-chromatin associations within individual nuclei. MUSIC presents a potent tool for exploring chromatin architecture and transcription at cellular resolution in complex tissues.


[STEPS]
SECTION: Crosslinking and nuclei isolation for cell lines
1. Wash cell culture with ice-cold PBS. 
SECTION: 5&#x27; Phosphorylation
36. Resuspend nuclei in 250 µL of 5’ phosphorylation master mix followed by an incubation at 37°C while rotating at 800 rpm for 1 hour.
SECTION: Ligation of cell barcodes
41. Anneal cell barcodes. 
SECTION: Ligation of cell barcodes
40. Aliquot up to 10w of cells for split-pool, and collect the rest of the cells to assess the DNA length after chromatin fragmentation.  
SECTION: Ligation of cell barcodes
46. The nuclei solutions from the 96 wells were pooled together into a 15 mL LoBind tube. 95 µL of quenching buffer was added to each well to rinse and collect any remaining nuclei and pooled into the same 15 mL tube.
SECTION: Ligation of cell barcodes
49. Repeat Steps 42-48 for the 2nd and 3rd rounds of split-pool.
SECTION: PolyA tailing
52. Incubate the nuclei at 37 ℃ for 10 min at 800 rpm in the PolyA Tailing Buffer. 
 

SECTION: PolyA tailing
53. Wash the nuclei once with 900 µL of PBS Wash Buffer 1 and three times with 900 µL of PBS Wash Buffer 2 at 900 x g for 2 min at 4°C.
SECTION: 10X GEM
57. Load the RT Mix to the 10X Chromium controller according to Steps 1.1 to 1.5 in the protocol of Chromium Next GEM Single Cell 3’ Reagent kit. Proceed to GEM generation (~18 min). 
 

SECTION: 10X GEM
58. Take out all emulsions (~120-150 µl) to a PCR tube. Incubate the GEM in Thermocycler using the following program: 
 

SECTION: 10X GEM
59. Recover nuclei acids form emulsion with 1:1 (~150 µl) of recovery reagent (Go to 10X protocol). Wait for 2 mins. Discard all organic (pink) phase.  Fill the aqueous phase to 200 µL with nuclease free water.
SECTION: Pre-PCR extension
63. Transfer 30 µl of the pre-PCR Master Mix into each of 8 PCR tubes. Transfer 20 µl of the eluted DNA into each of the 8 PCR tubes. Incubate eluted DNA at 55 ℃ for 15 min.
 

SECTION: Pre-PCR extension
64. Purify with 1.8X (90 µL) of RNA clean Ampure beads, and elute the sample with 20 µl of water.  
SECTION: Library size selection
67. Pool the 8 PCR elution, and load the elution into 5 wells of 4% E-gel. Check the size distribution.
SECTION: Crosslinking and nuclei isolation for brain tissues
16. Incubate samples with 10 mL of 2 mM disuccinimidyl glutarate (DSG) in 1X PBS in a 15 mL LoBind tube at room temperature for 45 min with gentle rotation. 
SECTION: Crosslinking and nuclei isolation for cell lines
4. Collect cell pellets by centrifugation at 330 x g for 3 min. 
SECTION: Crosslinking and nuclei isolation for cell lines
2. Incubate cells with 1 mL of Accutase (EMD, SF006) for 3 min at 37°C to dissociate the cells.
SECTION: Crosslinking and nuclei isolation for cell lines
3. Resuspend cells with 10 mL of ice-cold PBS to generate single cell suspension.
SECTION: Crosslinking and nuclei isolation for cell lines
5. Incubate cells with 10 mL of 2 mM disuccinimidyl glutarate (DSG) in 1X PBS in a 15 mL LoBind tube at room temperature for 45 min with gentle rotation. 
SECTION: Crosslinking and nuclei isolation for cell lines
6. Collect cells by centrifugation at 1,000 x g for 4 min, and wash cells once with 1X PBS. 
SECTION: Crosslinking and nuclei isolation for cell lines
7. Resuspend cells in 10 mL of 1X PBS containing 3% formaldehyde, and incubate cells for 10 min with a gentle rotation. 
SECTION: Crosslinking and nuclei isolation for cell lines
9. Wash cells twice with ice-cold 1X PBS containing 0.5% BSA (wt/vol), and centrifuge cells at 1,000 x g for 4 min.
SECTION: Crosslinking and nuclei isolation for cell lines
10. Resuspend cells in ice-cold 1X PBS with 0.5% BSA (wt/vol), and aliquot 5M cells into each 1.5 mL tube.
SECTION: Crosslinking and nuclei isolation for cell lines
11. Collect cells by centrifugation at 1,000 x g for 5 min and snap-freeze cells in liquid nitrogen, and stored at -80°C.
SECTION: Crosslinking and nuclei isolation for cell lines
12. Thaw frozen cells on ice, and incubate cells in 1.4 mL of cell lysis buffer A for every 5M cells for 10 min on ice.

 

SECTION: Crosslinking and nuclei isolation for cell lines
13. Collect cell pellets by centrifugation at 900 x g for 4 min at 4°C, and incubate cells in 1.4 mL of cell lysis buffer B on ice for 10 min.
 

SECTION: Crosslinking and nuclei isolation for cell lines
14. Centrifuge isolated nuclei at 900 x g for 5 min at 4°C.
SECTION: Crosslinking and nuclei isolation for brain tissues
15. Thaw 50 mg of post-mortem human brain frontal cortex sample on ice in a 1.5ml LoBind tube, and chop the tissue into smaller pieces by pestle.
SECTION: Crosslinking and nuclei isolation for brain tissues
17. Wash once with 10 mL of 1XPBS by centrifugation at 1,000 x g for 4 min.
SECTION: Crosslinking and nuclei isolation for brain tissues
18. Resuspend the sample in 15 mL of 1X PBS containing 3% formaldehyde, and incubate for 10 min with a gentle rotation.
SECTION: Crosslinking and nuclei isolation for brain tissues
19. Quench the crosslinking reaction by the addition of 5 mL of 1.25 M glycine followed by an incubation of 5 min with a rotation.
SECTION: Crosslinking and nuclei isolation for brain tissues
20. Centrifuge the sample at 1,000 x g for 4 min and washed twice with ice-cold 1X PBS containing 0.3% BSA (wt/vol).
SECTION: Crosslinking and nuclei isolation for brain tissues
21. Chromium Nuclei Isolation kit (10X genomics, 1000494) was used to isolate nuclei from crosslinked cortex samples according to the section for “single cell gene expression & chromium fixed RNA profiling” (page 25 to page 30 from sample user guide).
SECTION: Crosslinking and nuclei isolation for brain tissues
22. All the following steps (steps 23-71) are the same for either cell line or human cortex samples.
SECTION: Nuclei permeabilization
24. Add 60 µL of 1X rCutSmart buffer containing 10% Triton X-100 (wt/vol) into the SDS solution above, and incubate the reaction at 37°C for 15 min while shaking at 800 rpm.
SECTION: Nuclei permeabilization
23. Thoroughly resuspend and permeabilize the nuclei in 200 µL of 1X rCutSmart buffer (NEB, B7204S) containing 0.25% SDS at 62°C for 10 min using Eppendorf Thermomixer C (Eppendorf).
SECTION: Nuclei permeabilization
25. Wash treated nuclei once with 1X rCutSmart buffer by centrifugation at 900 x g for 2 min at 4°C.
SECTION: 5&#x27; Phosphorylation
26. Resuspend nuclei in 250 µL of 5’ Phosphorylation Master Mix followed by an incubation at 37°C while rotating at 800 rpm for 1 hour.
 

SECTION: 5&#x27; Phosphorylation
27. Wash the nuclei once with 900 µL of PBS Wash Buffer 1 and three times with 900 µL of PBS Wash Buffer 2 at 900 x g for 2 min at 4°C.
 

 

SECTION: dA-tailing
32. Incubate the cells at 37 ℃ for 1.5 h with rotation at 800 rpm in 250 µL of the dA-tailing mix. 
 

SECTION: RNA linker ligation
28. Incubate the isolated nuclei with 250 µL of the RNA Ligation Mix at 25ºC for 2 hours then 16ºC overnight with an intermittent mixing at 800 rpm (30 seconds on and 270 off).

 

SECTION: RNA linker ligation
29. Wash the nuclei once with 900 µL of PBS Wash Buffer 1 and three times with 900 µL of PBS Wash Buffer 2 at 900 x g for 2 min at 4°C.
SECTION: DNA linker ligation
34. Incubate the cells at 20 ℃ overnight with rotation at 800 rpm (30s on and 270s off) in 250 µL of the DNA linker ligation mix.
 



SECTION: dA-tailing
33. Wash the nuclei once with 900 µL of PBS Wash Buffer 1 and three times with 900 µL of PBS Wash Buffer 2 at 900 x g for 2 min at 4°C.
SECTION: Cell counting
38. Resuspend the nuclei in 900 µL of PBS Wash Buffer 2 with 0.2 U/µL of RNase Inhibitor, and filter the nuclei through a 10 µM cell strainer (pluriStrainer, 43-10010-50). 
SECTION: DNA linker ligation
35. Wash the nuclei once with 900 µL of PBS Wash Buffer 1 and three times with 900 µL of PBS Wash Buffer 2 at 900 x g for 2 min at 4°C.
SECTION: 5&#x27; Phosphorylation
37. Wash the nuclei once with 900 µL of PBS Wash Buffer 1 and three times with 900 µL of PBS Wash Buffer 2 at 900 x g for 2 min at 4°C.
SECTION: Cell counting
39. Stain 6 µL of the nuclei suspension with 6 µL of Ethidium homodimer-1, and count the number of nuclei by Countess II Automated Cell Counter (ThermoFisher). 
SECTION: Ligation of cell barcodes
41.1. Make 100 µl of 45 µM DNA oligo duplex plates, in each well of the PCR plate, the following reaction will occur (total 100 µL):
 
Annealing using hybridization program: 
Heat to 95°C and maintain the temperature for 2 min.
Cool to 22°C at a rate of -1°C/min.
Cool to 4 °C for temporary storage.
SECTION: Ligation of cell barcodes
41.2. Aliquot 2.4 µL of annealed oligo duplex into several new 96-well plates. Seal the plate and centrifuge at 800 g for 2 min.
SECTION: Ligation of cell barcodes
43. Pipette 6.4 µL of ligation master mix into each well of the 96-well plate above.
 

SECTION: 3&#x27; Dephosphorylation
50. Incubate the cells at 37 ℃ for 1h at 800 rpm in the 3’ Dephosphorylation Buffer.

 

 

SECTION: 3&#x27; Dephosphorylation
51. Wash the nuclei once with 900 µL of PBS Wash Buffer 1 and three times with 900 µL of PBS Wash Buffer 2 at 900 x g for 2 min at 4°C.
SECTION: Sonication
54. Filter nuclei in PBS+0.04% BSA via a 10 µM filter.
SECTION: Sonication
55. Stain 6 µL of the nuclei suspension with 6 µL of Ethidium homodimer-1, and count the number of nuclei by Countess II Automated Cell Counter (ThermoFisher). 
SECTION: Sonication
56. Transfer 5k cells to a Covaris microtube-15, and filled the nuclei suspension to 15 µL with 1X PBS with 0.04% BSA (wt/vol). Sonicate the nuclei using Covaris M220 Focused-ultrasonicator with water temperature 6ºC, incident power 50 W, duty factor 5 for 5 min.
SECTION: Reverse crosslinking
60. Aliquot 25 µl of the recovered nuclei solution into each of 8 LoBind 1.5 mL tubes with 25 µl of 2X reverse crosslinking mix to each of the 8 tubes. 
 

SECTION: Reverse crosslinking
61. Incubate the tubes at 50 ℃ for 2h at 800 rpm (30s on and 270s off) and 55 ℃ overnight at 800 rpm (30s on and 270s off).
SECTION: Library amplification
65. Conduct PCR for 13 cycles using the Ultra 2 program: 
 

SECTION: Library amplification
66. Purify the library with 1.2X (60 µL) of RNA clean Ampure beads. Elute with 12.5 µL of water. 
SECTION: Reverse crosslinking
62. Purify the nuclei acid in 8 tubes with NEB RNA clean up kit (NEB, 76307-460) and elute in 21 µl of water.  
SECTION: Quantity/quality check and sequencing
69. Check the quantity with Qubit.
SECTION: Quantity/quality check and sequencing
70. Check the size distribution with Tape station.   
SECTION: Quantity/quality check and sequencing
71. Sequence the library. Set the Read 1 of the sequencer to 28 bp, the Index 1 to 8 bp, and the Read 2 to 150 bp.
SECTION: Crosslinking and nuclei isolation for cell lines
8. Quench the reaction by adding 3 mL of 2.5 M glycine per 10 mL of 3% formaldehyde and incubating cells for 5 min with rotation.
SECTION: Chromatin digestion
30. Resuspend the nuclei in a Digestion Master Mix followed by an incubation at 37°C for 3 h while rotating at 800 rpm. 
 

SECTION: Chromatin digestion
31. Wash the nuclei once with 900 µL of PBS Wash Buffer 1 and three times with 900 µL of PBS Wash Buffer 2 at 900 x g for 2 min at 4°C.
SECTION: Ligation of cell barcodes
42. Fill the nuclei suspension to 1144 µL with PBS Wash Buffer 2 and 24 µL of RNAse inhibitor, and pipette 11.2 µL of cell solution into each well of the 96-well plate that contains cell barcodes. 
SECTION: Ligation of cell barcodes
44. Ligate Barcode Set 1 with the linkers in each well in the Ligation Master Mix at 20ºC overnight with an intermittent mixing at 1600 rpm (30 seconds on and 270 off). 
SECTION: Ligation of cell barcodes
45. Quench the ligation by an incubation for 10 min at 20ºC at 1600 rpm (30s on and 270s off) in 60 µL of quenching buffer for each well of the 96-well plate. 
SECTION: Ligation of cell barcodes
47. Centrifuge the pooled nuclei at 900 x g for 4 min, and transfer the nuclei into a 1.5 mL tube with 0.5 mL of remaining supernatant. Rinse the 15 mL tube with 500 µL of PBS Wash Buffer 2, and collect the residual nuclei into the same 1.5 mL tube. 
SECTION: Ligation of cell barcodes
48. Wash the nuclei three times with 900 µL of PBS Wash Buffer 2 by the centrifugation at 900 x g for 2 min. 
SECTION: Library size selection
68. Excise bands from 300-1200 bp. Extract the DNA with NEB Monarch gel purification kit (NEB, T1020S) using two columns, and elute the DNA with 15 µL of the elution buffer for each column.