Jun 03, 2024

Public workspaceMUSIC Protocol V.2

DOI
dx.doi.org/10.17504/protocols.io.4r3l22kwxl1y/v2
  • 1Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: Jeff.spraggins@vanderbilt.edu
Wenxin Zhao
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DOI: dx.doi.org/10.17504/protocols.io.4r3l22kwxl1y/v2
Protocol Citation: Wenxin Zhao, Zhifei Luo, Sheng Zhong 2024. MUSIC Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l22kwxl1y/v2Version created by Wenxin Zhao
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: February 20, 2024
Last Modified: June 03, 2024
Protocol Integer ID: 101151
Funders Acknowledgements:
NIH grant
Grant ID: DP1DK126138
NIH grant
Grant ID: R01GM138852
NIH grant
Grant ID: UH3CA256960
NIH grant
Grant ID: U01CA200147
NIH grant
Grant ID: R01HD107206
Kruger Research Grant
Abstract
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.

Crosslinking and nuclei isolation for cell lines
Crosslinking and nuclei isolation for cell lines
Wash cell culture with ice-cold PBS.
Incubate cells with 1 mL of Accutase (EMD, SF006) for 3 min at 37°C to dissociate the cells.
Resuspend cells with 10 mL of ice-cold PBS to generate single cell suspension.
Collect cell pellets by centrifugation at 330 x g for 3 min.
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.
Collect cells by centrifugation at 1,000 x g for 4 min, and wash cells once with 1X PBS.
Resuspend cells in 10 mL of 1X PBS containing 3% formaldehyde, and incubate cells for 10 min with a gentle rotation.
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.
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.
Resuspend cells in ice-cold 1X PBS with 0.5% BSA (wt/vol), and aliquot 5M cells into each 1.5 mL tube.
Collect cells by centrifugation at 1,000 x g for 5 min and snap-freeze cells in liquid nitrogen, and stored at -80°C.
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.

ABCD
ComponentWorking Conc.Stock Conc.Volume
HEPES, pH 7.450 mM1 M2.5 mL
NaCl140 mM5 M1.4 mL
EDTA, pH 8.01 mM0.5 M100 uL
EGTA, pH 8.01 mM0.5 M100 uL
Glycerol 10%100%5 mL
Triton X-1000.25%100%625 uL
NP-400.5%100%250 uL
Water 10.475 mL
Total Volume50 mL
Cell lysis buffer A

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.
ABCD
ComponentWorking Conc.Stock Conc.Volume
Tris-HCl, pH 810 mM1 M0.5 mL
NaCl200 mM5 M2 mL
EDTA, pH 8.01.5 mM0.5 M150 uL
EGTA, pH 8.01.5 mM0.5 M150 uL
Water 47.2 mL
Total Volume50 mL
Cell lysis buffer B

Centrifuge isolated nuclei at 900 x g for 5 min at 4°C.
Crosslinking and nuclei isolation for brain tissues
Crosslinking and nuclei isolation for brain tissues
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.
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.
Wash once with 10 mL of 1XPBS by centrifugation at 1,000 x g for 4 min.
Resuspend the sample in 15 mL of 1X PBS containing 3% formaldehyde, and incubate for 10 min with a gentle rotation.
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.
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).
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).
All the following steps (steps 23-71) are the same for either cell line or human cortex samples.
Nuclei permeabilization
Nuclei permeabilization
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).
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.
Wash treated nuclei once with 1X rCutSmart buffer by centrifugation at 900 x g for 2 min at 4°C.
5' Phosphorylation
5' Phosphorylation
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.
ABC
ReagentFInal Conc.Volume (uL)
10X T4PNK buffer1X25
T4PNK (10 U/uL)0.5 U/uL12.5
ATP (10 mM)1 mM25
RNasin (40 U/uL)1 U/uL6.25
Water181.25
Total Volume250
5’ Phosphorylation Master Mix

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.
ABCD
ComponentWorking Conc.Stock Conc.Volume
PBS, pH 7.51X10X5 mL
EDTA, pH 8.01 mM0.5 M100 uL
EGTA, pH 8.01 mM0.5 M100 uL
Triton X-1000.1%100%50 uL
Water 44.75 mL
Total Volume50 mL
PBS Wash Buffer 1

ABCD
ComponentWorking Conc.Stock Conc.Volume
PBS, pH 7.51X10X5 mL
Triton X-1000.1%100%50 uL
BSA0.3%10%1.5 mL
Water 43.45 mL
Total Volume50 mL
PBS Wash Buffer 2

RNA linker ligation
RNA linker ligation
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).

ABC
ReagentFInal Conc.Volume (uL)
10X T4 RNA ligation buffer1X25
T4 RNA ligase 1 (10 U/uL)0.4 U/uL10
PEG (50%)15 %75
ATP (10 mM)1 mM25
RNasin (40 U/uL)1 U/uL6.25
100 uM RNA linker4 uM10
DMSO18.75
Water80
Total Volume250
RNA Ligation Mix

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.
Chromatin digestion
Chromatin digestion
Resuspend the nuclei in a Digestion Master Mix followed by an incubation at 37°C for 3 h while rotating at 800 rpm.
ABC
ReagentFInal Conc.Volume (uL)
10X rCutSmart buffer1X30
HpyCH4V (5 U/uL)0.5 U/uL30
RNasin (40 U/uL)1 U/uL7.5
Water232.5
Total Volume300
Chromatin Digestion Master Mix

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.
dA-tailing
dA-tailing
Incubate the cells at 37 ℃ for 1.5 h with rotation at 800 rpm in 250 µL of the dA-tailing mix.
ABC
ReagentFInal Conc.Volume (uL)
10X NEBNext dA-Tailing reaction buffer1X25
Klenow fragment (5 U/uL)0.2 U/uL10
RNasin (40 U/uL)1 U/uL6.25
Water208.75
Total Volume250
dA-Tailing mix

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.
DNA linker ligation
DNA linker ligation
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.
ABC
ReagentFInal Conc.Volume (uL)
NEBNext Quick Ligation Reaction Buffer (5X)0.8X40
Instant Sticky-end Ligase Master Mix (2X)0.2X25
1, 2-Propanediol (100%)6%15
DNA linker (45 uM)4.5 uM25
RNasin (40 U/uL)1 U/uL6.25
Water138.75
Total Volume250
DNA linker ligation mix

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.
5' Phosphorylation
5' Phosphorylation
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.
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.
Cell counting
Cell counting
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).
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).
Ligation of cell barcodes
Ligation of cell barcodes
Aliquot up to 10w of cells for split-pool, and collect the rest of the cells to assess the DNA length after chromatin fragmentation.
Anneal cell barcodes.
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):
ABCD
ComponentStock Conc.Working Conc.Volume (uL)
Top strand oligo100 uM45 uM45
Bottom strand oligo100 uM45 uM45
NaCl5 M50 mM1
ddH2O9
Total Volume100
Oligo Annealing
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.
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.
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.
Pipette 6.4 µL of ligation master mix into each well of the 96-well plate above.
ABC
ReagentFInal Conc.Volume (uL)
NEBNext Quick Ligation Reaction Buffer (5X)0.8X960
Instant Sticky-end Ligase Master Mix (2X)0.2X600
1, 2-Propanediol (100%)6%360
Total Volume1920
Ligation master mix

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).
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.

ABCD
ComponentWorking Conc.Stock Conc.Volume
PBS, pH 7.51X10X5 mL
EDTA, pH 8.050 mM0.5 M5 mL
EGTA, pH 8.050 mM0.5 M5 mL
Triton X-1000.1%100%50 uL
Water 34.95 mL
Total Volume50 mL
Quenching buffer

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.
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.
Wash the nuclei three times with 900 µL of PBS Wash Buffer 2 by the centrifugation at 900 x g for 2 min.
Repeat Steps 42-48 for the 2nd and 3rd rounds of split-pool.
3' Dephosphorylation
3' Dephosphorylation
Incubate the cells at 37 ℃ for 1h at 800 rpm in the 3’ Dephosphorylation Buffer.

ABCD
ComponentStock Conc.Working Conc.Volume (uL)
Tris-HCl, pH 6.51 M350 mM350
MgCl21 M10 mM50
DTT100 mM10 mM100
Water500
Total Volume1000
5X PNK Buffer

ABC
ReagentFInal Conc.Volume (uL)
5X PNK Buffer1X50
T4PNK (10 U/uL)0.5 U/uL12.5
RNasin (40 U/uL)1 U/uL6.25
Water181.25
Total Volume250
3’ Dephosphorylation Buffer

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.
PolyA tailing
PolyA tailing
Incubate the nuclei at 37 ℃ for 10 min at 800 rpm in the PolyA Tailing Buffer.
ABC
ReagentFInal Conc.Volume (uL)
10X E.coli Poly(A) Polymerase Reaction Buffer1X25
E.coli Poly(A) Polymerase (5 U/uL)0.08 U/uL4
ATP (10 mM)1 mM25
RNasin (40 U/uL)1 U/uL6.25
Water189.75
Total Volume250
PolyA Tailing Buffer

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.
Sonication
Sonication
Filter nuclei in PBS+0.04% BSA via a 10 µM filter.
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).
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.
10X GEM
10X GEM
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).
AB
ReagentVolume (uL)
RT Reagent B18.8
Reducing Agent B2
RT Enzyme C8.7
Sonicated nuclei20
Water20.5
Total Volume70
RT Mix

Take out all emulsions (~120-150 µl) to a PCR tube. Incubate the GEM in Thermocycler using the following program:
AB
Temp. (°C)Time
5345 min
855 min
Slowly cool down at 0.1°C/s15 min
4Hold
Thermocycler Program for Reverse Transcription

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.
Reverse crosslinking
Reverse crosslinking
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.
ABC
ReagentFInal Conc.Volume (uL)
NaCl (5 M)0.4 M20
SDS (20%)o.4%5
EDTA (0.5 M)50 mM25
EGTA (0.5 M)50 mM25
Proteinase K (0.8 U/uL)0.04 U/uL12.5
Water162.5
Total Volume250
2X Reverse Crosslinking Master Mix

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).
Purify the nuclei acid in 8 tubes with NEB RNA clean up kit (NEB, T2050S) and elute in 21 µl of water.
Pre-PCR extension
Pre-PCR extension
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.
ABC
ReagentFInal Conc.Volume (uL)
10X Isothermal Amplification Buffer II1X5
Bst 3.0 DNA polymerase (8 U/uL)0.32 U/uL2
MgSO4 (100 mM)6 mM3
dNTP Mix (10 mM)1.4 mM * 47
RNasin (40 U/uL)0.5 U/uL0.625
Water12.375
Total Volume30
pre-PCR Master Mix

Purify with 1.8X (90 µL) of RNAClean XP beads, and elute the sample with 20 µl of water.
Library amplification
Library amplification
Conduct PCR for 13 cycles using the Ultra 2 program:
ABC
ReagentFInal Conc.Volume (uL)
NEBNext Q5 Mater Mix1X25
Illumina Universal Adaptor (10 uM)0.5 uM2.5
Illumina Index Adaptor 1-8 (10 uM)0.5 uM2.5
Sample20
Total Volume50
PCR Master Mix

Purify the library with 1.2X (60 µL) of AMPure XP beads. Elute with 12.5 µL of water.
Library size selection
Library size selection
Pool the 8 PCR elution, and load the elution into 5 wells of 4% E-gel. Check the size distribution.
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.
Quantity/quality check and sequencing
Quantity/quality check and sequencing
Check the quantity with Qubit.
Check the size distribution with Tape station.
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.