Protocol for investigating chromatin architecture in adult skeletal muscle stem cells using high-throughput chromosome conformation capture

Summary

3D genome architecture is pivotal for gene transcription and cellular lineage development. However, it remains challenging to generate high-quality Hi-C (high-throughput chromosome conformation capture) libraries from rare cell populations. Here, we present a protocol for investigating chromatin architecture in adult skeletal muscle stem cells, also known as satellite cells (MuSCs), using Hi-C. We detail the procedures for isolating MuSCs, provide an in-depth guide for preparing Hi-C libraries, and introduce the use of the Microcket package for processing Hi-C data.

For complete details on the use and execution of this protocol, please refer to Zhao et al.¹ and Zhao et al.²

Graphical abstract

Highlights

• •Guidance on isolation of adult skeletal muscle stem cells
• •Step-by-step protocol for generating Hi-C libraries from 40,000 to 500,000 MuSCs
• •Instructions for Hi-C data processing using Microcket

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

3D genome architecture is pivotal for gene transcription and cellular lineage development. However, it remains challenging to generate high-quality Hi-C (high-throughput chromosome conformation capture) libraries from rare cell populations. Here, we present a protocol for investigating chromatin architecture in adult skeletal muscle stem cells, also known as satellite cells (MuSCs), using Hi-C. We detail the procedures for isolating MuSCs, provide an in-depth guide for preparing Hi-C libraries, and introduce the use of the Microcket package for processing Hi-C data.

Before you begin

Skeletal muscle has a remarkable regenerative ability, largely owing to the resident muscle stem cells, named satellite cells (MuSCs).³ These cells predominantly reside in a quiescent state in the physiological niche, thus serving as a good model for studying stem cell quiescence and chronological aging.⁴ However, it remains challenging to study the molecular events in MuSCs, as they represent a rare cell population and the isolation yield is normally low. For instance, profiling genome-wide chromatin interactions using conventional Hi-C protocols^5,6,7 recommends a starting cell number over 1 million, which normally exceeds the MuSC yield from one mouse.⁸ Moreover, the number of MuSCs declines with aging and in certain pathological conditions.⁹ Here, we present a modified Hi-C protocol to generate high-quality, reproducible Hi-C libraries from 40,000-500,000 MuSCs isolated from one mouse. In this protocol, we describe two alternative approaches to isolate MuSCs from the transgenic Pax7-nGFP mice¹⁰: one for obtaining the freshly isolated SCs (FISCs) and one for quiescent SCs (QSCs). We discriminate these two conditions, as we and others have found they are different in transcriptomes, histone modifications and 3D genome architecture.^1,11,12,13 To obtain QSCs, the step of in situ fixation by paraformaldehyde before fluorescence-activated cell sorting (FACS) isolation is required, which has been proven to preserve the quiescence status of SCs.^1,11 Following the isolation step, we provide a step-to-step guide to generate Hi-C libraries from either FISCs or QSCs, including crosslinking of SCs, chromosome conformation capture and library preparation for Illumina sequencing. In this section, we present several adaptations to original in situ Hi-C protocols, allowing us to use a reduced amount of staring material. At the end, we provide an outline for Hi-C data processing using our Microcket pipeline,² an ultra-fast, sensitive, and versatile toolkit for the alignment of sequencing reads in Hi-C data.

The protocol below describes the specific steps for generating Hi-C libraries from MuSCs. Generally, we isolate 400,000-600,000 FISCs or 100,000-200,000 QSCs from a Pax7-nGFP transgenic mouse aged 2-3 months, which is enough for one Hi-C library preparation. We have also applied this protocol in MuSCs isolated from wild-type C57BL/6 mice of different ages, as well as in other cell types (e.g., C2C12 myoblast, HeLa-S3 and K562 cell line). Theoretically, this protocol holds the potential for studying 3D genome architecture in other rare cell populations.

We have made several adaptations to the original in situ Hi-C protocols,^5,6 including modifications to the quantities of enzymes, biotinylated nucleotides, and the cell lysis steps, to accommodate the low amount of MuSCs. Therefore, we recommend a starting cell number less than 1 million when applying this protocol. For more starting material, one can split the sample into multiple tubes with 1-million cells as an aliquot or stick to the original in situ Hi-C protocols.

Institutional permissions

All mice experiments were performed under the approval of the Institutional Animal Care and Use Committee (IACUC) from Sun Yat-sen University or Animal Experimentation Ethics Committee (AEEC) of the Chinese University of Hong Kong (CUHK). Researchers utilizing this protocol are required to acquire authorization and training from their institution’s Animal Ethics Committee.

Isolation of in situ-fixed adult skeletal muscle stem cells

Timing: 1 day

• 1.
  Dissection and mechanical dissociation of hind limb muscles.

  • a.
    Prepare 10-cm petri dishes with 10 mL of PBS, 10-cm glass dishes with 10 mL of fixative solution and 50-ml tubes containing 30 mL of fixative solution.

    Note: Place all dishes and tubes on ice to chill the fixative solution.

  • b.
    Euthanize the mice by cervical dislocation. Spray the mouse with 70% (vol/vol) ethanol, and place it in the dissection area.
  • c.
    Lift the skin with forceps and cut a small incision with scissors. Extend the incision, and expose all the hind limb muscles.
  • d.
    Dissect the hind limb muscles in the petri dish with 10 mL of PBS. Trim away visible fat tissue and tendon.
  • e.
    Quickly transfer the muscle pieces in the glass dishes containing 10 mL of fixative solution. Slice the muscle with razor blades ∼10 times to break the fascia and expose the muscle to the fixative solution.

    CRITICAL: Rapid exposure of the muscle pieces to the fixative solution is essential for maintaining MuSCs in a quiescent state.

  • f.
    Dip the muscle pieces into the 50-ml tube containing 30 mL of ice-cold fixation solution.
  • g.
    Repeat steps e-f to collect all hind limb muscles.

    Note: The total dissection time does not exceed 15 min.

  • h.
    Decant the muscle pieces with fixative solution in a fresh glass dish. Carefully slice the muscle with razor blades for ∼10 min. The obtained muscle pieces range 1 and 5 mm.

    Note: Ensure that the muscle pieces remain immersed in the fixation solution.

• 2.
  Fixation of muscles.

  • a.
    Transfer minced muscle with fixative solution into a new 50-ml tube.
  • b.
    Gently rotate at a rotating mixer for 1 h at 4°C.
  • c.
    Centrifuge at 900 g for 5 min at 4°C. Discard the supernatant.
  • d.
    Fill each tube to 50 mL with ice-cold PBS and gently invert to resuspend the pellet.
  • e.
    Gently rotate for 15 min at 4°C.
  • f.
    Centrifuge at 900 g for 5 min at 4°C. Discard the supernatant.
  • g.
    Repeat steps d-f two more times to remove residual fixation buffer.
  • h.
    Transfer all muscles of a mouse to a fresh petri dish containing 10 mL of QSC dissociation buffer. Carefully slice the muscle with razor blades for ∼10 min.
  • i.
    Transfer minced muscle with QSC dissociation buffer into a 50-ml tube.
• 3.
  Enzyme digestion and isolation of mononucleated cells.

  • a.
    Seal the tubes with parafilm and incubate at 37°C in a shaking water bath at 75 rpm for 1.5 h.

    Note: Ensure the tubes are fully submerged in water during incubation.

  • b.
    Fill each tube to 50 mL with cold wash medium and gently invert to mix.
  • c.
    Centrifuge at 900 g for 5 min at 4°C. Aspirate the supernatant to ∼15 mL.
  • d.
    Add 2 mL of QSC collagenase II solution and 2 mL of QSC dispase solution.
  • e.
    Resuspend the pellet by gently pipetting up and down 10 times with a 5-ml pipette.
  • f.
    Seal the tubes with parafilm and incubate at 37°C in a shaking water bath at 75 rpm for 30 min.

    Note: Ensure the tubes are fully submerged in water during incubation.

  • g.
    Aspirate and eject the muscle suspension 15 times using a 10-ml syringe with a 21-gauge needle. See troubleshooting, problem 1.
  • h.
    Fill each tube to 50 mL with cold wash medium and gently invert to mix.
  • i.
    Centrifuge at 900 g for 5 min at 4°C. Aspirate the supernatant to ∼10 mL.
  • j.
    Resuspend the pellet with a 10-ml pipette. Filter the supernatant through a 40-μm nylon cell strainer into a fresh 50-ml tube.
  • k.
    Wash the original tube with 10 mL of cold wash medium and filter through the same cell strainer.
  • l.
    Rinse the cell strainer with another 10 mL of wash medium.
  • m.
    Fill each tube to 50 mL with wash medium and gently invert to mix.
  • n.
    Centrifuge at 900 g for 5 min at 4°C. Immediately aspirate the supernatant, being very careful not to disturb the pellet.
  • o.
    Resuspend the cell pellet in 1 mL of wash medium.
• 4.
  Cell sorting.

  • a.
    Set up the cell sorter with the 70-μm nozzle following the manufacturer’s instructions (https://www.bdbiosciences.com/content/dam/bdb/marketing-documents/BD_FACSAria_III_User_Guide.pdf).
  • b.
    Run the cells at the lowest flow rate to verify the voltages are appropriate and the cell population is properly positioned in the FSC-A and SSC plot.
  • c.
    Create a gate on the FSC-A and SSC-A plot that excludes debris (Figure 1A).
  • d.
    Create a gate on the FSC-H plot and SSC-H plots to collect intact single cells (Figure 1A).
  • e.
    Create a gate to collect the GFP+ population (P4, Figure 1A) into 7 mL of wash medium in a 15-ml tube.
  • f.
    Centrifuge at 900 g for 5 min at 4°C. Discard supernatant.

    Note: Do not try to remove all of the supernatant. Leave ∼20 μL supernatant is fine.

  • g.
    Proceed immediately to step “crosslinking of MuSCs”.

Figure 1.

Representative gating strategy to isolate MuSCs from Tg: Pax7-nGFP mice by FACS

Mononucleated cells were isolated from limb muscles of 2-month-old Tg: Pax7-nGFP mice.

(A) Representative FACS profiles of in situ fixed SCs (QSC). The yield of QSCs that we obtain represents ∼1–2% of the total mononucleated cells.

(B) Representative FACS profiles of freshly isolated SCs (FISC). The yield of FISCs that we obtain represents ∼4–5% of the total mononucleated cells.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Nutrient mixture F-10 Ham	Sigma-Aldrich	Cat# N6635
Penicillin-streptomycin mixture	Gibco	Cat# 15140122
Horse serum, heat inactivated, New Zealand origin	Gibco	Cat# 26050088
Collagenase, type 2	Worthington Biochemical	Cat# LS004176
Dispase	Gibco	Cat# 17105041
PBS, pH 7.4	Gibco	Cat# 10010023
Formaldehyde solution	Sigma-Aldrich	Cat# 47608
Glycine	Diamond	Cat# A100167
UltraPure 1 M Tris-HCI, pH 8.0	Invitrogen	Cat# 15568025
NaCl (5 M), RNase-free	Invitrogen	Cat# AM9760G
IGEPAL CA-630	Sigma-Aldrich	Cat# I8896
cOmplete, EDTA-free protease inhibitor cocktail	Roche	Cat# 04693132001
SDS	BBI	Cat# A600485
Triton X-100	Sigma-Aldrich	Cat# T8787
MgCl2 (1 M)	Invitrogen	Cat# AM9530G
DpnII	New England Biolabs	Cat# R0543L
NEBuffer 3.1	New England Biolabs	Cat# B7203
dCTP	Takara	Cat# 4028
dGTP	Takara	Cat# 4027
dTTP	Takara	Cat# 4029
Biotin-14-dATP	Invitrogen	Cat# 19524016
DNA polymerase I, large (Klenow) fragment	New England Biolabs	Cat# M0210L
T4 DNA ligase reaction buffer	New England Biolabs	Cat# B0202S
T4 DNA ligase	New England Biolabs	Cat# M0202L
BSA	New England Biolabs	Cat# B9000S
EDTA (0.5 M), pH 8.0, RNase-free	Invitrogen	Cat# AM9260G
Tween 20	Sigma-Aldrich	Cat# P9416
Proteinase K	New England Biolabs	Cat# P8107S
UltraPure phenol:chloroform:isoamyl alcohol (25:24:1, v/v)	Invitrogen	Cat# 15593031
Glycogen (5 mg/mL)	Invitrogen	Cat# AM9510
TE buffer, 1×, molecular biology grade	Promega	Cat# V6231
Agarose	Biowest	Cat# 111860
50× TAE	Biosharp	Cat# BL533A
10× loading buffer	Takara	Cat# 9157
SuperGreen nucleic acid gel stain	Biosharp	Cat# BS355A
Trans2K Plus II DNA marker	TransGen Biotech	Cat# BM121-01
Dynabeads MyOne streptavidin C1	Invitrogen	Cat# 65001
VAHTS DNA clean beads	Vazyme	Cat# N411
Critical commercial assays
NEBNext Ultra II DNA library prep kit for Illumina	New England Biolabs	Cat# E7645S
VAHTS DNA adapters set 1 for Illumina	Vazyme	Cat# N801
VAHTS DNA adapters set 2 for Illumina	Vazyme	Cat# N802
Experimental models: Organisms/strains
Mouse: B6.Cg-Tg(Pax7-EGFP)15Tajb/J (female, 2 month old)	The Jackson Laboratory	RRID:IMSR_JAX:036759
Software and algorithms
Microcket	Zhao et al., 20242	https://github.com/hellosunking/Microcket
Other
Forceps with straight tips	RWD	Cat# F11011-11
Dissection scissors	RWD	Cat# S12003-09
Sterile surgical blade size 11	RWD	Cat# S31011-01
Petri dish, 10 cm	Fisher Scientific	Cat# FB0875713
Filter unit	Millipore	Cat# SLGPR33RB
50-mL centrifuge tubes	NEST	Cat# 602002
15-mL centrifuge tubes	NEST	Cat# 601002
5-mL serological pipette	NEST	Cat# 326001
10-mL serological pipette	NEST	Cat# 327001
1.5 mL Eppendorf tube	Axygen	Cat# MCT-175-C
DNA LoBind tube 1.5 mL	Eppendorf	Cat# 022431021
Syringes, 10 mL	BD Biosciences	Cat# 302995
21-gauge needles	BD Biosciences	Cat# 305166
Falcon 40-μm cell strainer	Corning	Cat# 352340
Falcon 5-mL round-bottom tubes with a strainer cap	Corning	Cat# 352235
Phase Lock Gel-Heavy	Tiangen Biotech	Cat# WM5-2302830
Four-dimensional rotating mixer	Kylin-Bell Lab Instruments	Cat# BE-1100
Shaking water bath	LABOAO	Cat# LH-110∗12
Refrigerated centrifuge with swing rotor	Eppendorf	Cat# 5810R
Refrigerated microcentrifuge	Eppendorf	Cat# 5418
FACSAria cytometer	BD Biosciences	BD FACSAria III Cell Sorter
Eppendorf ThermoMixer C	Eppendorf	Cat# 5382000023
NanoDrop One spectrophotometer	Thermo Fisher Scientific	Cat# 840-317500
microTUBE AFA fiber with snap cap	Covaris	Cat# 500045
S220 focused ultrasonicator	Covaris	Cat# 500217
Magna GrIP rack	Millipore	Cat# 20-400
PCR machine (T100 thermal cycler)	Bio-Rad	Cat# 1861096
Gel imaging system	Tanon	Cat# 1600
4150 TapeStation system	Agilent Technologies	Cat# G2992AA
Qubit 3.0 fluorometer	Thermo Fisher Scientific	Cat# Q33216

Materials and equipment

Wash medium

Ham’s F-10 supplemented with 10% (vol/vol) horse serum and 1× penicillin-streptomycin. The wash medium could be stored at 4°C for up to 1 month.

FISC dissociation buffer

Dissolve collagenase II powder in wash medium so that the final concentration is 1,000 U/ml. Prepare freshly just before the dissection and keep on ice until needed. Prepare 10 mL for each mouse.

FISC collagenase II solution

Dissolve collagenase II powder in 1× PBS so that the final concentration is 1,000 U/ml. Store in 1-ml aliquots at −20°C for up to 3 months.

FISC dispase solution

Dissolve dispase powder in 1× PBS so that the final concentration is 11 U/ml. Store in 1-ml aliquots at −20°C for up to 3 months.

4% (w/v) paraformaldehyde solution

Add 40 g of paraformaldehyde to 800 mL PBS. Stir the mixture at 60˚C and add 5 N NaOH dropwise until a clear solution is formed. Adjust the volume to 1 L. Sterilize with a 0.22-μm filter. Adjust pH with small amounts of 1 N HCl to approximately 6.9. The fixative solution could be stored at 4°C for up to one month.

CRITICAL: Paraformaldehyde is toxic. NaOH and HCl are highly corrosive. HCl can cause severe skin burns. Wear the laboratory coat, gloves and goggles when handling.

Fixative solution

Mix 62.5 mL 4% (w/v) paraformaldehyde solution with 437.5 mL 1 × PBS so that the final concentration is 0.5%.

QSC dissociation buffer

Dissolve collagenase II powder in wash medium so that the final concentration is 2,000 U/ml. Prepare freshly just before the dissection and keep on ice until needed. Prepare 10 mL for each mouse.

QSC collagenase II solution

Dissolve collagenase II powder in 1× PBS so that the final concentration is 2,000 U/ml. Store in 1-ml aliquots at −20°C for up to 3 months.

QSC dispase solution

Dissolve dispase powder in 1× PBS so that the final concentration is 22 U/ml. Store in 1-ml aliquots at −20°C for up to 3 months.

1% (w/v) formaldehyde solution

Mix 27 μL of 37% (w/v) formaldehyde with 973 μL of 1 × PBS. Prepare freshly just before use.

CRITICAL: Formaldehyde is toxic. Wear the laboratory coat, gloves and goggles when handling it.

2.5 M glycine

Dissolve 3.7535 g of glycine in 15 mL of Milli-Q water. Adjust the volume to 20 mL. Sterilize with a 0.22-μm filter and store at 20°C–25°C for several months.

10% (w/v) SDS

Dissolve 5 g of SDS in 40 mL of Milli-Q water. Adjust the volume to 50 mL. Sterilize with a 0.22-μm filter and store at 20°C–25°C for several months.

CRITICAL: SDS can cause skin irritation and eye damage. Wear the laboratory coat, gloves, and goggles when handing it.

10% (vol/vol) Triton X-100

Mix 1 mL of 100% (vol/vol) Triton X-100 with 9 mL of Milli-Q water. The solution could be stored at 4 °C for 6 months.

10% (vol/vol) IGEPAL CA630

Mix 1 mL of 100% (vol/vol) IGEPAL CA630 with 9 mL of Milli-Q water. The solution could be stored at 4°C for 6 months.

10 mM Tris-Cl (pH 8.0)

Mix 10 μL of 1 M Tris-Cl (pH 8.0) with 990 μL of Milli-Q water. Prepare freshly just before use.

0.1× TE

Mix 1× TE with 9 mL of Milli-Q water. The solution could be stored at 4°C for 6 months.

Hi-C lysis buffer

Reagent	Final concentration	Amount
Tris-HCl (pH 8.0) (1 M)	10 mM	10 μL
NaCl (5 M)	10 mM	2 μL
Igepal CA630 (10%)	0.2%	20 μL
Proteinase inhibitor cocktail (100 ×)	1 ×	10 μL
Milli-Q H2O	N/A	958 μL
Total	N/A	1 mL

Note: Prepare freshly just before use and keep on ice until needed to ensure efficient lysis.

1.25× NEB buffer 3.1

Reagent	Final concentration (1.25×)	Amount
Tris-HCl (pH 8.0) (1 M)	62.5 mM	62.5 μL
NaCl (5 M)	125 mM	25 μL
MgCl2 (1 M)	12.5 mM	12.5 μL
BSA (10 mg/mL)	0.125 mg/mL	12.5 μL
Milli-Q H2O	N/A	887.5 μL
Total	N/A	1 mL

Note: Prepare freshly just before use. This protocol utilizes the DpnII enzyme for chromatin digestion. When using a different restriction enzyme, adjust the reaction conditions (including reaction buffer, temperature, and digestion time) accordingly.

Biotin fill-in mix

Reagent	Final concentration	Amount
NEBuffer 3.1 (10 ×)	1 ×	5 μL
dGTP (1 mM)	30 μM	1.5 μL
dCTP (1 mM)	30 μM	1.5 μL
dTTP (1 mM)	30 μM	1.5 μL
Biotin-14-dATP (0.4 mM)	30 μM	3.75 μL
Klenow Fragment (5 U/μL)	0.2 U/μL	2 μL
Milli-Q H2O	N/A	34.75 μL
Total	N/A	50 μL

Note: Prepare freshly just before use. When using a restriction enzyme other than DpnII, different biotinylated dNTPs may be required.

Alternatives: We have also tested the use of Biotin-14-dATP (1 mM, Jena Bioscience, Cat# NU-835-BIO14-L), DNA Polymerase I, Large (Klenow) Fragment (5 U/μL, Abclonal, Cat# RK20525) during the biotin fill-in step.

Ligation mix

Reagent	Final concentration	Amount
T4 DNA Ligase Buffer (10 ×)	1 ×	50 μL
BSA (10 mg/mL)	0.1 mg/mL	5 μL
T4 DNA Ligase (400 U/μL)	0.8 U/μL	1 μL
Milli-Q H2O	N/A	394 μL
Total	N/A	450 μL

Note: Prepare freshly just before use.

Alternatives: In addition to T4 DNA Ligase (NEB, Cat# M0202L), we have tested the use of T4 DNA Ligase (Abclonal, Cat# RK21500).

1× Tween wash buffer (TWB)

Reagent	Final concentration	Amount
Tris-HCl (pH 8.0) (1 M)	5 mM	50 μL
EDTA (0.5 M)	0.5 mM	10 μL
NaCl (5 M)	1 M	2 mL
Tween-20 (10%)	0.05%	50 μL
Milli-Q H2O	N/A	7.89 mL
Total	N/A	10 mL

Note: Sterilize with a 0.22-μm filter. The 1× TWB buffer could be stored at 4°C for 6 months.

2× binding buffer (BB)

Reagent	Final concentration	Amount
Tris-HCl (pH 8.0) (1 M)	10 mM	100 μL
EDTA (0.5 M)	1 mM	20 μL
NaCl (5 M)	2 M	4 mL
Milli-Q H2O	N/A	5.88 mL
Total	N/A	10 mL

Note: Sterilize with a 0.22-μm filter. 2× BB buffer could be stored at 4°C for 6 months.

Step-by-step method details

Isolation of freshly isolated adult skeletal muscle stem cells

Timing: 1 day

During this step, freshly isolated SCs (FISCs) are obtained from a 2-month-old female Pax7-nGFP mice. The procedures involve dissection of hind limb muscles, isolation of mononucleated cells, and cell sorting. Additionally, an alternative procedure for isolation of in situ fixed adult skeletal muscle stem cells (QSCs) is outlined in the “before you begin” section. Following this step, it is essential for the FISCs to proceed promptly to the next stage, “crosslinking of MuSCs”.

• 1.
  Dissection and mechanical dissociation of hind limb muscles.

  • a.
    Prepare 10-cm petri dishes with 10 mL of PBS (one dish per mouse).
  • b.
    Euthanize the mice by cervical dislocation. Spray the mouse with 70% (vol/vol) ethanol, and place it in the dissection area.
  • c.
    Lift the skin with forceps and cut a small incision with scissors. Extend the incision, and expose all the hind limb muscles.
  • d.
    Dissect the hind limb muscles in the petri dish. Trim away visible fat tissue and tendons.
  • e.
    Transfer all muscles of a mouse to a fresh glass dish containing 10 mL of FISC dissociation buffer.
  • f.
    Using forceps to hold one end of a piece of muscle, carefully slice the muscle with razor blades for ∼10 min. See troubleshooting, problem 2.

    CRITICAL: At the end of this step, the muscle tissue should be optimally minced, as shown in Figure 2. Under-mincing or over-mincing can result in a low yield of MuSCs.

  • g.
    Transfer minced muscle with FISC dissociation buffer into a 50-mL tube.
• 2.
  Enzyme digestion and isolation of mononucleated cells.

  • a.
    Seal the tubes with parafilm and incubate at 37°C in a shaking water bath at 75 rpm for 1.5 h.

    Note: Ensure the tubes are fully submerged in water during incubation.

  • b.
    Fill each tube to 50 mL with cold wash medium and gently invert to mix.
  • c.
    Centrifuge at 700 g for 5 min at 4°C. Aspirate the supernatant to ∼15 mL.
  • d.
    Add 2 mL of FISC collagenase II solution and 2 mL of FISC dispase solution.
  • e.
    Resuspend the pellet by gently pipetting up and down 10 times with a 5-ml pipette.
  • f.
    Seal the tubes with parafilm and incubate at 37°C in a shaking water bath at 75 rpm for 30 min.

    Note: Ensure the tubes are fully submerged in water during incubation.

  • g.
    Aspirate and eject the suspension 15 times using a 10-ml syringe with a 21-gauge needle.
  • h.
    Fill each tube to 50 mL with cold wash medium and gently invert to mix.
  • i.
    Centrifuge at 700 g for 5 min at 4°C. Aspirate the supernatant to ∼10 mL.
  • j.
    Resuspend the pellet with a 10-ml pipette. Filter the supernatant through a 40-μm nylon cell strainer into a fresh 50-ml tube.
  • k.
    Wash the original tube with 10 mL of cold wash medium and filter through the same cell strainer.
  • l.
    Rinse the cell strainer with another 10 mL of wash medium.
  • m.
    Fill each tube to 50 mL with cold wash medium and gently invert to mix.
  • n.
    Centrifuge at 700 g for 5 min at 4°C. Immediately aspirate the supernatant, being very careful not to disturb the pellet.
  • o.
    Resuspend the cell pellet in 1 mL of wash medium.
• 3.
  Cell sorting.

  • a.
    Set up the cell sorter with the 70-μm nozzle following the manufacturer’s instructions (https://www.bdbiosciences.com/content/dam/bdb/marketing-documents/BD_FACSAria_III_User_Guide.pdf).
  • b.
    Run the cells at the lowest flow rate to verify the voltages are appropriate and the cell population is properly positioned in the FSC-A and SSC plot.
  • c.
    Create a gate on the FSC-A and SSC-A plot that excludes debris (Figure 1B).
  • d.
    Create a gate on the FSC-H plot and SSC-H plots to collect intact single cells (Figure 1B).
  • e.
    Create a gate to collect the GFP+ population (P4, Figure 1B) into 7 mL of wash medium in a 15-ml tube.
  • f.
    Centrifuge at 700 g for 5 min at 4°C. Discard supernatant.

    Note: Do not try to remove all of the supernatant if proceeding directly to the crosslinking step. Leave ∼20 μL supernatant is fine.

  • g.
    Proceed immediately to step “crosslinking of MuSCs”.

    Note: Alternatively, collected FISCs can be cultured by seeding cells onto coated culture dishes.

Figure 2.

Representative images of under-, optimally, and over-minced muscle samples

Crosslinking of MuSCs

Timing: 40 min

In this step, formaldehyde is used to crosslink FISCs or QSCs. Formaldehyde is a commonly employed agent in Hi-C experiments, as it reacts with both proteins and DNA, thereby preserving chromatin interactions. After this step, the fixed MuSCs can either proceed to the cell lysis step or be snap-frozen and stored at −80°C.

• 4.Prepare 1% formaldehyde solution by adding 27 μL of 37% formaldehyde to 973 μL of PBS. Mix by vortexing the tube several times.

CRITICAL: In the following steps, use fresh, intact and RNase-/DNase-free tubes and pipette tips. Avoid using autoclaved tubes and tips, as this may lead to cell adhesion to the sides of the tubes or tips during the centrifugation process, resulting in significant cell loss.

• 5.Resuspend the cell pellet in 100 μL of 1% formaldehyde solution. Using a 100-200 μL pipette, gently and thoroughly pipette the mixture 15-20 times. Add the remaining 900 μL of formaldehyde solution.

Note: We recommend first resuspend the cell pellet thoroughly in a small volume (100 μL) with a 100-200 μL pipette, followed by the addition of the remaining buffer.

• 6.Incubate at 20°C–25°C for 10 min without rotating. Pipette up and down for several times at the incubation midpoint.

Note: Rotating on a rocker is not recommended when working with a low number of MuSCs, which may lead to cell loss.

• 7.Quench the crosslink reaction by adding 87 μL of 2.5 M glycine to the tube to a final concentration of 0.2 M. Pipette up and down to mix thoroughly.
• 8.Incubate at 20°C–25°C for 5 min without rotating.
• 9.Centrifuge at 900 g for 5 min at 4°C. Discard supernatant. See troubleshooting, problem 3.
• 10.Resuspend cells in 1 mL of cold 1× PBS and centrifuge at 900 g for 5 min at 4°C. Discard supernatant. See troubleshooting, problem 3.

Note: It is important to completely remove the liquid at this step. If the pellet appears loose or adheres to the side of tube, re-centrifuge once at 2,500 g.

• 11.Snap-freeze the cell pellet in liquid nitrogen and stored at −80°C or one can continue with cell lysis.

Pause Point: The cell pellets can be stored at −80°C for up to one year.

Chromosome conformation capture

Timing: 3–4 days

During this step, cross-linked MuSCs are subjected to the downstream Hi-C procedures, which include cell lysis, chromatin digestion, biotinylation of DNA ends, proximity ligation, cross-link reversal, and DNA purification. Compared with the original in situ Hi-C protocols, we adjust the quantities of enzymes, biotinylated nucleotides, and the cell lysis steps to accommodate the low number of MuSC population. We have successfully generated Hi-C libraries using approximately 40,000 to 500,000 MuSCs isolated from one mouse. After this step, the purified DNA can be stored at −20°C prior to the DNA shearing step.

• 12.
  Cell lysis.

  • a.
    Prepare 1 mL Hi-C lysis buffer and chill the buffer on ice prior to use.

    Note: If the cell pellets have been stored in −80°C, thaw them on ice before proceeding. For the number of MuSCs less than 1 million, we typically use 1 mL lysis buffer. Scale up the volume if the cell number is increased or multiple samples are processed simultaneously.

  • b.
    Resuspend the cell pellet in 100 μL of ice-cold Hi-C lysis buffer. Using a 100–200 μL pipette, gently and thoroughly pipette the mixture 15–20 times. Add an additional 400 μL of ice-cold Hi-C lysis buffer.

    Note: We recommend first resuspend the cell pellet thoroughly in a small volume (100 μL) with a 100–200 μL pipette, followed by the addition of the remaining buffer.

  • c.
    Incubate cell suspension on ice for 20 min. Pipette up and down for several times at the incubation midpoint.

    Note: Rotating on a rocker is not recommended when working with a low number of MuSCs, which may lead to cell loss.

  • d.
    Centrifuge at 2,500 g for 5 min at 4°C. Discard the supernatant.

    Note: If the pellet appears loose or adheres to the side of tube, re-centrifuge once at 5,000 g. For efficient buffer exchange, first remove ∼90% of the supernatant, then re-centrifuge the tube while maintaining the same orientation, and carefully remove the remaining supernatant without disturbing the pellet. This applies to all following steps involving supernatant removal after centrifugation.

  • e.
    Repeat the lysis procedure once by following steps b-d.
• 13.
  Chromatin digestion.

  • a.
    Prepare 1.25× NEB Buffer 3.1.
  • b.
    Wash the pelleted nuclei once with 500 μL of 1.25× NEB Buffer 3.1. Centrifuge at 2,500 g for 5 min and discard the supernatant.

    Note: It is not necessary to resuspend the cell pellet at this step. It may lead to cell loss.

  • c.
    Gently resuspend the pellet in 355 μL of 1.25 × NEB Buffer 3.1.
  • d.
    Add 11 μL of 10% SDS (final concentration: 0.3% SDS) and mix carefully by pipetting up and down. Avoid making bubbles.
  • e.
    Incubate the mixture at 37°C for 1 h with shaking at 950 rpm in a Thermomixer. See troubleshooting, problem 4.
  • f.
    Add 75 μL of 10% Triton X-100 and mix carefully by pipetting up and down. Incubate at 37°C for 1 h with shaking at 950 rpm in a Thermomixer to quench the SDS. See troubleshooting, problem 4.
  • g.
    Add 10 μL of DpnII enzyme (10 U/μl, 100 U in total) and mix gently. Incubate at 37°C for 12-16 h with shaking at 950 rpm in a Thermomixer. See troubleshooting, problem 4.

    Note: The incubation period can be extended to 16–18 hours if necessary.

• 14.
  Biotin fill-in.

  • a.
    Incubate the reaction solution at 62°C for 20 min to inactivate DpnII, then cool to 20°C–25°C.
  • b.
    During the incubation, prepare the biotin fill-in mix.
  • c.
    After incubation (step a), centrifuge at 2,500 g for 5 min. Discard as much supernatant as possible.
  • d.
    Add 50 μL of the biotin fill-in mix. Mix gently by pipetting up and down. Avoid making bubbles.
  • e.
    Incubate the tubes at 23°C for 4 h in a Thermomixer (950 rpm mixing; 15 secs every 1 min).
• 15.
  Proximity ligation.

  • a.
    By the end of the biotin fill-in incubation (step 14), prepare the ligation mix.
  • b.
    Add 450 μL of the ligation mix to the fill-in reaction. Mix by pipetting up and down for several times.
  • c.
    Incubate at 20°C–25°C for 4 h or longer (12–16 h is fine) with slow rotation or incubate in a Thermomixer with interval shaking (950 rpm mixing; 15 secs every 1 min).

Note: At this stage, chromatin has now been covalently ligated.

• 16.
  Cross-linking reversal.

  • a.
    Pellet nuclei by centrifuge at 2,500 g for 5 min. Discard 380 μL of the supernatant.
  • b.
    Add 12 μL of 10% SDS. Mix by pipetting up and down for several times. Avoid making bubbles.
  • c.
    Add 5 μL of 20 mg/ml proteinase K. Mix by pipetting up and down for several times.
  • d.
    Incubate at 55°C for 2 h in a Thermomixer with shaking at 1,000 rpm.
  • e.
    Add 13 μL of 5 M NaCl and mix gently by pipetting up and down. Incubate at 65°C for 12–16 h.

Note: Reverse crosslinking could be performed for a minimum of 6 h.

• 17.
  DNA purification.

  • a.
    Cool the tube to 20°C–25°C. Add 150 μL of H2O, then add 300 μL phenol: chloroform: isoamyl alcohol (P:C:IA). Vortex the mixture for 30-60 s and transfer the mixture to a 2 mL Phase Lock Gel-Heavy.
  • b.
    Centrifuge the mixture at 12,000 g for 10 min at 20°C–25°C.
  • c.
    Transfer the top aqueous layer 300 μL to a new 1.5 mL tube containing 4 μL of glycogen (5 μg/μL).
  • d.
    Add 750 μL of 100% ethanol (vol/vol) and vortex thoroughly. Incubate the mixture for 30 min at −80°C.

    Note: The sample will become viscous during this step.

  • e.
    Centrifuge the mixture at 14,000 g for 20 min at 4°C.
  • f.
    Remove the supernatant and wash the pellet once in 80% (vol/vol) ethanol.
  • g.
    Air-dry the DNA samples for ∼10 min or until visibly dry.
  • h.
    Re-dissolve the DNA pellet in 130 μL 10 mM Tris-Cl, pH 8.0.
  • i.
    Incubate at 37°C for 15 min to fully dissolve the DNA.
  • j.
    Take 1 μL DNA sample and measure the concentration using Nanodrop One Spectrophotometer.

    Pause Point: The purified DNA can be stored at −20°C indefinitely prior to the DNA shearing step.

Hi-C library preparation

Timing: 1–2 days

During this step, proximity-ligated DNA is fragmented by sonication, followed by biotinylated fragment enrichment with streptavidin beads. The resulting DNA is then converted into a Hi-C library suitable for Illumina paired-end sequencing. Compared with original in situ Hi-C protocols,^5,6 we include a test PCR amplification step to determine the optimal number of PCR cycles for the full-scale PCR reaction.

• 18.
  DNA shearing and Biotin pull-down.

  • a.
    Shear the DNA to a size range of 300-500 bp using a Covaris S220 or Covaris M220 with the following parameters.

    Parameters	Covaris S220	Covaris M220
    Peak incident power (W)	175	75
    Duty cycle/duty factor	10%	20%
    Cycles per burst	200	200
    Treatment time (s)	150	120

    Note: While various sonicators can be used (e.g., Bioruptor), parameters must be optimized to achieve the DNA fragment size range of 300-500 bp. See troubleshooting, problem 5.

  • b.
    Transfer the sheared DNA to a fresh 1.5 mL tube. Wash the Covaris tube with 20 μL of water and add to the sample, bringing the total volume to 150 μL.
  • c.
    Wash 10 μL of 10 mg/ml Dynabeads MyOne Streptavidin C1 beads with 100 μL of 1× Tween Washing Buffer (TWB). Separate the beads on a magnet and discard the supernatant.

    Note: Use 2 μl MyOne Streptavidin C1 beads per 1 μg Hi-C DNA, with a minimum of 10 μl of beads.

  • d.
    Resuspend the beads in 150 μL of 2× Binding Buffer (BB) and add to the DNA sample.
  • e.
    Incubate at 20°C–25°C for 30 min. Mix by pipetting up and down for several times at the incubation midpoint.
  • f.
    Place the tube on a magnetic stand to reclaim the beads. Wait until the solution is clear (∼1 min), and carefully discard the supernatant.
  • g.
    Wash the beads by adding 100 μL of 1×TWB. Heat the tubes on a Thermomixer at 55°C for 2 min with mixing at 1,000 rpm.
  • h.
    Reclaim the beads using a magnetic stand. Wait until the solution becomes clear (∼1 min), and carefully discard the supernatant.
  • i.
    Repeat the TWB wash by following steps g-h.
  • j.
    Wash the beads by adding 50 μL of 10 mM Tris-Cl (pH 8.0) and transfer the mixture to a new 1.5 mL tube.
  • k.
    Reclaim the beads using a magnetic stand and discard the supernatant.
  • l.
    Resuspend the beads with 50 μL of 10 mM Tris-Cl (pH 8.0) and transfer the mixture to a 0.2 mL PCR tube.
• 19.
  End repair and adaptor ligation.

  • a.
    Add the following components to the 0.2 mL PCR tube containing the DNA sample.

    End repair reaction

    Reagent	Amount
    NEBNext Ultra II End Prep Reaction Buffer	7 μL
    NEBNext Ultra II End Prep Enzyme Mix	3 μL

    Note: Library preparation was performed on-bead using components from the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Cat# E7645S).

    Alternatives: Equivalent DNA library preparation kits from other suppliers may also be suitable and should be evaluated by the user. We have also utilized the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, Cat# ND607) to generate Hi-C libraries, achieving comparable library yields.

  • b.
    Gently pipette the entire volume up and down at least 10 times to mix thoroughly. If necessary, perform a quick spin to collect all liquid from the sides of the tube.
  • c.
    Place the tube in a thermocycler, with the heated lid set to 80°C. Incubate at 20°C for 30 min, followed by incubation at 65°C for 30 min. Keep the sample at 4°C until the next step.
  • d.
    Add the following components directly to the End Prep Reaction Mixture.

    Reagent	Amount
    Indexed Adaptor (1:20 diluted from 15 μM stock)	2.5 μL
    NEBNext Ligation Enhancer	1 μL
    NEBNext Ultra II Ligation Master Mix	30 μL

    Note: The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time. We used VAHTS DNA Adapters Set 1 for Illumina (Vazyme, Cat# N801) and VAHTS DNA Adapters Set 2 for Illumina (Vazyme, Cat# N802) in adaptor ligation step. Equivalent adaptors from other suppliers may also be suitable and should be evaluated by the user.

  • e.
    Pipette the entire volume up and down at least 10 times to mix thoroughly. If necessary, perform a quick spin to collect all liquid from the sides of the tube.

    Note: The NEBNext Ultra II Ligation Master Mix is highly viscous. Do not vortex while mixing the ligation reaction.

  • f.
    Incubate at 20°C for 15 min in a thermocycler with the heated lid off.
  • g.
    Transfer the mixture to a new 1.5 mL tube. Place the tube on a magnetic stand to reclaim the beads. Wait until the solution is clear (∼1 min), and carefully discard the supernatant.
  • h.
    Wash the beads by adding 100 μL of 1× Tween Washing Buffer (TWB). Heat the tubes on a Thermomixer at 55°C for 2 min with mixing at 1,000 rpm.
  • i.
    Reclaim the beads using a magnetic stand. Wait until the solution becomes clear (∼1 min), and carefully discard the supernatant.
  • j.
    Repeat the TWB wash by following steps h-i.
  • k.
    Wash the beads by adding 50 μL of 10 mM Tris-Cl (pH 8.0) and transfer the mixture to a new 1.5 mL tube. Reclaim the beads using a magnetic stand and discard the supernatant.
  • l.
    Resuspend the beads with 27 μL of 10 mM Tris-Cl (pH 8.0) and transfer the mixture to a 0.2 mL PCR tube.
  • m.
    Incubate at 98°C for 10 min in a thermocycler. Quickly separate on a magnetic stand and transfer 26 μL of the solution to a new 0.2 mL PCR tube.
• 20.
  PCR cycle number optimization (test PCR amplification).

  • a.
    Prepare a series of 4-fold dilutions of the DNA sample in 10 mM Tris-Cl (pH 8.0). Test PCR amplification, consisting a total of 20 cycles, will be conducted on these dilutions to determine the optimal number of PCR cycles for full-scale amplification.

    Test PCR amplification

    Dilution	Amount of DNA	Amount of ddH2O	Equivalent full-scale PCR cycles
    1	2 μL of DNA sample	6 μL	16
    2	2 μL of Dilution 1	6 μL	14
    3	2 μL of Dilution 2	6 μL	12
    4	2 μL of Dilution 3	6 μL	10

    Note: Since only 6 μL of the total 8 μL from Dilution 1 is utilized for PCR, this corresponds to an equivalent of 1.5 μL (6/8 × 2) of the original DNA sample, representing a 16-fold less than the remaining 24 μL DNA sample. Therefore, the equivalent number of PCR cycles is 16 in the full-scale amplification, relative to the test PCR of Dilution 1. Each subsequent dilution in the series will require an additional reduction of 2 cycles compared to the preceding dilution, resulting in 14, 12, and 10 cycles for Dilutions 2–4, respectively.

  • b.
    Prepare a test PCR mix. One test PCR mix will be used for each dilution prepared in step a. For processing multiple samples, scale up accordingly.

    Test PCR mix

    Reagent	Amount
    10× Library Amplification Primer mix	1.5 μL
    NEBNext Ultra II Q5 Master Mix	7.5 μL

  • c.
    Add 9 μL of the PCR mix to 6 μL of each diluted test sample (dilutions 1–4).
  • d.
    Perform test PCR amplification using the following thermal cycling conditions.

    PCR cycling conditions

    Steps	Temperature	Time	Cycles
    Initial denaturation	98 °C	30 s	1
    Denaturation	98 °C	10 s	20
    Annealing/Extension	65 °C	75 s
    Final Extension	65 °C	5 min	1
    Hold	4 °C	∞

  • e.
    Add 2 μL of 10× Loading buffer to the 15 μL PCR reactions, and load 17 μL of the samples onto a 1% agarose gel in 1× TAE.
  • f.
    Run the gel at 120 V for 35 min.
  • g.
    Image the gel with a 470-nm blue light transilluminators or UV light. Analyze the gel to identify the dilution corresponding to the appropriate full-scale PCR amplification cycle (Figure 3). See troubleshooting, problems 6 and 7.

    Note: To determine the optimal number of PCR cycles, we routinely select the lowest number of cycles that yields a visible smear on gel.

• 21.
  Full-scale PCR amplification.

  • a.
    Add 36 μL of the PCR mix to the rest 24 μL of DNA sample.

    Full-scale PCR mix

    Reagent	Amount
    10× Library Amplification Primer mix	6 μL
    NEBNext Ultra II Q5 Master Mix	30 μL

  • b.
    Use the following thermal cycling to perform full-scale PCR amplification.

    PCR cycling conditions

    Steps	Temperature	Time	Cycles
    Initial denaturation	98 °C	30 s	1
    Denaturation	98 °C	10 s	optimized cycle
    Annealing/Extension	65 °C	75 s
    Final Extension	65 °C	5 min	1
    Hold	4 °C	∞

• 22.
  Clean up the PCR products.

  • a.
    During the full-scale PCR amplification step, take out the VAHTS DNA Clean Beads from 4°C to equilibrate to 20°C–25°C for ∼30 min. See troubleshooting, problem 6.

    Alternatives: Equivalent DNA cleanup products from other suppliers, such as AMPure XP Beads (Beckman Coulter, Cat# A63881), SPRIselect Beads (Beckman Coulter, Cat# B23318), are also be suitable.

  • b.
    Vortex the VAHTS DNA Clean Beads thoroughly to ensure homogeneity. Add 45 μL (∼0.75×) of resuspended beads to the 60 μL PCR reaction.
  • c.
    Mix thoroughly by pipetting up and down at least 10 times.
  • d.
    Incubate the mixture on bench top for at least 5 min at 20°C–25°C.
  • e.
    Place the PCR tube on a magnetic stand to reclaim the beads. Wait until the solution is clear (∼5 min), then carefully remove the supernatant.
  • f.
    While keeping the beads on the magnet, wash once with 200 μL of fresh 80% ethanol without mixing. Incubate at 20°C–25°C for 30 sec, then carefully remove and discard the supernatant.
  • g.
    Repeat 80% ethanol wash once by following step f.
  • h.
    Spin the tube, place back on the magnetic stand and remove any residual ethanol using a 10-μL pipette tip.
  • i.
    Air-dry the beads for up to 5 min while the tube is on the magnetic stand with the lid open.

    CRITICAL: Avoid over-drying the beads, as this may lead to reduced recovery of the DNA. Elute the samples while the beads are still dark brown and glossy, but all visible liquid has evaporated. If the beads turn lighter brown and begin to crack, they are too dry.

  • j.
    Add 15-20 μL of 0.1× TE buffer to elute DNA library. Mix by pipetting, incubate at 20°C–25°C for 5 min, separate on a magnet, and transfer the solution to a fresh 1.5 mL tube.
  • k.
    Quantify the DNA content of Hi-C library using either fluorometry-based (e.g., Qubit, PicoGreen) or qPCR-based methods, and determine the size distribution of Hi-C library by an Agilent TapeStation (Figure 4) or Bioanalyzer.

Figure 3.

Gel images of Hi-C library products

(A) Hi-C condition optimization. 1 million C2C12 cells were used to generate Hi-C library. Following the test amplification stage, PCR products at four dilutions were analyzed on the agarose gel. From the left, the dilutions correspond to 16, 14, 12 and 10 cycles of full amplification. The lane labeled ‘Beads’ represents the PCR reaction using the beads after heat treatment (98°C for 10 min) as the template. The absence of PCR products indicates efficient release of Hi-C DNA from the beads into the aqueous solution.

(B) Representative MuSCs Hi-C libraries at the test amplification stage. In the first sample, PCR products at four dilutions, corresponding to 16, 14, 12 and 10 cycles of full amplification, were analyzed on the agarose gel. The other three different samples were analyzed at three dilutions, corresponding to 16, 14 and 12 cycles. We routinely select the lowest number of cycles that yields a visible smear on gel, as the optimal amplification cycles (12 cycles for these examples).

Figure 4.

Size distribution of MuSCs Hi-C libraries

The average size for the two Hi-C libraries is 462 bp and 458 bp respectively, as determined with the High Sensitivity D1000 ScreenTape. FU, fluorescent unit.

Sequencing and Hi-C data analysis

Timing: 10 days

In this step, we recommend using Microcket,² an ultra-fast, sensitive, and versatile toolkit for the alignment of sequencing reads in Hi-C data. Alternatively, one can use other widely used tools (e.g., Juicer¹⁴ and HiC-Pro¹⁵) for processing Hi-C data.

• 23.Sequence Hi-C libraries on the Illumina HiSeq X Ten platform or Illumina NovaSeq X Plus platform using 150 bp paired-end mode.

Note: Sequencing depth is a critical factor in resolving 3D genome structures at different scales. Generally, analyses at the compartmental level do not require high sequencing depth, with 30–40 million reads (∼20–25 million valid pairs) being sufficient. In contrast, finer-scale structures such as topologically associating domains (TADs) and chromatin loops demand much more reads. For instance, we routinely sequence over 1 billion reads for MuSCs to achieve loop-level resolution.

• 24.Download the latest version of Microcket² (currently v.1.4) from GitHub.

> wget https://github.com/hellosunking/Microcket/archive/refs/tags/v1.4.tar.gz-O Microcket.v1.4.tar.gz

> tar zxf Microcket.v1.4.tar.gz

## you will see a new directory named “Microcket-1.4”, go into it

> cd Microcket-1.4

• 25.Build genome indices.

Note: Here we utilize the mouse reference genome NCBI GRCm38 (UCSC mm10) for aligning sequencing reads in Hi-C data derived from MuSCs. You only need to build the index once for each reference genome.

> wget https://hgdownload.soe.ucsc.edu/goldenPath/mm10/bigZips/latest/mm10.fa.gz

## remove the unplaced contigs (optional but recommended)

> perl util/clean.genome.pl mm10.fa.gz >mm10.clean.fa

## build index and generate annotation files

> ./util/build.index.sh mm10.clean.fa mm10 bwa

• 26.Create a file containing the paths to your fastq files.

Note: Here we use our previous Hi-C data as an example: QSC Hi-C (GEO accession number: GSM5708470), which contains 3 replicates with the following accessions: SRR17068908, SRR17068909, and SRR17068910; FISC (GEO accession number: GSM5708476), which contains 2 replicates with the following accessions: SRR17068893 and SRR17068894.

## create a directory for this testing purpose

> mkdir test.data

> cd test.data

## download and decompress the data

> for sid in SRR17068908 SRR17068909 SRR17068910 SRR17068893 SRR17068894

> do

> wget https://sra-pub-run-odp.s3.amazonaws.com/sra/$sid/$sid -O $sid.sra

> fasterq-dump $sid.sra

> done

## record the path of the fastq files

> for sid in SRR17068908 SRR17068909 SRR17068910

> do

> echo -e "$PWD/${sid}_1.fastq∖t$PWD/${sid}_2.fastq"

> done > GSM5708470.fq.list

> for sid in SRR17068893 SRR17068894

> do

> echo -e "$PWD/${sid}_1.fastq∖t$PWD/${sid}_2.fastq"

> done > GSM5708476.fq.list

• 27.Run Microcket on the data using 32 threads. The resulting “.hic” file can be visualized with Juicebox software and for downstream analyses. See troubleshooting, problem 8.

> ../microcket -a BWA -g mm10 -x -t 32 -i GSM5708470.fq.list -o GSM5708470

> ../microcket -a BWA -g mm10 -x -t 32 -i GSM5708476.fq.list -o GSM5708476

Note: Microcket reports alignment results in both “pairs” and “hic” formats. In this instance, the output files would be “GSM5708470.hic” and “GSM5708476.hic”. In addition, Microcket provides the statistics during the alignment, as shown in Table 1. For downstream TAD identification, the “pairs” and “hic” format files can be directly utilized with various TAD callers, such as Arrowhead,⁵ IS,¹⁶ and Topdom.¹⁷ It is important to note that different tools may yield varying results regarding TAD sizes, numbers, and subTAD identification.^18,19,20 For downstream chromatin loop identification, the “pairs” and “hic” format files can be applied using tools such as FitHic2,²¹ HiCCUPS,⁵ or Mustache.²²

Table 1.

Basic statistics of in situ Hi-C data for QSC and FISC

	QSC (GSM5708470)		FISC (GSM5708476)
	Count	Fraction (%)	Count	Fraction (%)
Total	189,395,485	100.0	136,332,826	100.0
Ktrim preprocessed	187,461,440	99.0	135,024,445	99.0
Unique reads	157,824,527	84.2	109,353,338	81.0
Stitched reads	112,085,642	71.0	74,977,346	68.6
Unstitched reads	45,738,866	29.0	34,375,991	31.4
Discarded (too-short)	19	0.0	1	0.0
Mappable reads	147,425,564	93.4	102,221,081	93.5
Uncalled reads	10,159,657	6.9	6,869,841	6.7
Incomplete-mapping	8,910,855	6.0	6,115,688	6.0
Too-many-segments	542,081	0.4	313,483	0.3
Unpairable	703,773	0.5	439,090	0.4
Self-circle	2,948	0.0	1,580	0.0
Reported alignments	137,265,907	93.1	95,351,240	93.3
Cis (<1K)	25,852,991	17.5	16,523,315	16.2
Cis (1-10K)	7,449,334	5.1	4,232,826	4.1
Cis (>=10K)	80,562,195	54.6	53,834,900	52.7
Trans	23,401,387	15.9	20,760,199	20.3
Running time (h)	2.46		1.74

Expected outcomes

The protocol describes how to prepare Hi-C libraries from MuSCs. In our hands, PCR cycle number optimization step (step 20) is critical and provides a way to assess the quality of Hi-C libraries. Figure 3 represents the expected length distribution and PCR cycle titration results on the agarose gel. When attempting Hi-C library preparation for the first time, we recommend using a cell line with a comparable amount of staring material, such as 1 million C2C12 myoblasts, to evaluate the efficiency of this protocol. The Hi-C library DNA typically exhibits a smear on the agarose gel ranging from 200 to 700 bp (Figure 3). The absence of adaptor dimers at ∼150 bp and a low number of PCR cycles needed to obtain sufficient Hi-C DNA product, generally indicate high-quality libraries. To determine the optimal amplification cycles, we routinely select the lowest number of cycles that yields a visible smear on the gel (Figures 3B and 12 cycles for these examples). When applying this protocol on MuSCs, starting with a cell count of 40,000 to 500,000, 12 PCR cycles are typically sufficient for library amplification. Exceeding 14 PCR cycles is not recommended for deep sequencing, as we note this leads to an excessive amount of PCR duplicates in the sequencing reads. Figure 4 presents the length distribution of Hi-C libraries analyzed by Tapestation. The average size of Hi-C libraries is ∼460 bp.

A typical result of Hi-C libraries generated from MuSCs following this protocol includes > 50% of the reads are uniquely mapped with more than 75% of these being intra-chromosomal contacts (cis). Table 1 presents the alignment statistics of two Hi-C libraries from MuSCs reported by Microcket.² For these libraries, the uniquely mapped rates were 72.5% for QSC (GEO accession number: GSM5708470) and 70% for FISC (GEO accession number: GSM5708476), respectively. Inter-chromosomal contacts (trans) account for < 20% and more than 50% of unique reads are long-range intra-chromosomal contacts (Cis, >=10K).

Figure 5 presents an example of Hi-C contact maps generated from QSC and FISC. Various genome organization features can be identified from Hi-C contact matrix at different scales, including compartments, topologically associating domains (TADs) and chromatin loops.

Figure 5.

Representative Hi-C contact maps generated from QSC and FISC

Hi-C interaction maps showing a 50 Mb region of chr16 at 100-kb resolution (left), a 10 Mb region at 25-kb resolution (middle), and a 1.5 Mb region at 5-kb resolution (right).

Limitations

We have tested this protocol primarily on MuSCs isolated from Pax7-nGFP transgenic mice and wild-type C57BL/6 mice of different ages, as well as on cultured MuSCs.¹ In addition, we successfully applied this protocol on C2C12 myoblasts and some human cell lines including HeLa-S3 and K562. However, this protocol has not yet been evaluated on other primary cells or cell lines. We anticipate that it may also be applicable to other rare cell populations.

Recently, the Hi-C procedures have been revisited to assess the effects of different fixation methods and restriction enzymes on final data quality.²³ The up-to-date variants of Hi-C protocols may incorporate disuccinimidyl glutarate (DSG) in addition to formaldehyde fixation and use a combination of different restriction enzymes or MNase to digest chromatin.⁷ However, we have not yet tested for other fixation methods or restriction enzymes in this protocol.

Troubleshooting

Problem 1

The needles are easily clogged when isolating QSCs (step 3 of Isolation of in situ fixed adult skeletal muscle stem cells (QSCs) section).

Potential solution

When the muscle tissues have been fixed in paraformaldehyde, pieces of tendon or undigested muscle are evident after the enzyme digestion. The needles may be easily clogged. If clogging occurs during aspiration, carefully remove the obstruction from the tip of the needle. If clogging occurs during ejection, move the plunger up and down to dislodge the blockage.

Problem 2

Muscles are over-minced or under-minced, leading to the poor MuSCs yield (step 1).

Potential solution

Determine the optimal mincing conditions during the first attempts to isolate MuSCs. We typically use razor blades to slice the muscle. Over-mincing will release MuSCs into the suspension and leads to potential cell loss during centrifugation. Conversely, under-mincing may result in incomplete digestion, which can hinder the release of MuSCs into the suspension.

Problem 3

Cells adhere to the side of the tubes or float in the supernatant after centrifugation (steps 9-10).

Potential solution

Increase the centrifugation speed to 2,500 g. If cells still adhere to the side of the tubes, add 0.05% (w/v) of BSA or 0.01% (v/v) of IGEPAL CA630 to the PBS before re-centrifugation.

Problem 4

Evident cell clumps form in 1.5-ml tubes (step 13).

Potential solution

Resuspend the cell pellet sufficiently and gently at each step. In our hands, the solution should be homogenous and cell clumps are barely visible during chromatin digestion step.

Problem 5

Optimizing DNA shearing conditions with other sonicators (step 18).

Potential solution

To fragment DNA using a Bioruptor Pico (Diagenode, Cat# B01080010), we recommend the following protocol as a starting point: 100 μL DNA sample in 0.65 mL Bioruptor Microtubes (Diagenode, Cat# WA-005-0500); 30 sec ON/90 sec OFF for 3 cycles; Quick spin and pipet to mix; 30 sec ON/90 sec OFF for 3 cycles (for a total of 6 cycles). Check the size distribution of 2 μl of the fragmented DNA on a TapeStation or Bioanalyzer. If the DNA fragments are larger than 500 bp, sonicate those samples for additional 2–3 cycles of 30 sec ON/90 sec OFF. For other types of sonicators, we recommend first consulting the instrument documentation for DNA shearing and then performing the necessary optimization.

Problem 6

Prominent DNA band at ∼150 bp on the agarose gel (step 20).

Potential solution

• •The possible reason is adaptor dimers form during PCR step.
• •Reduce the amount of indexed adaptor used in step 19 or perform one round of VAHTS DNA Clean Beads purification before PCR step.
• •During PCR cleanup (step 22), perform two consecutive rounds of DNA cleanup.

Problem 7

No visible DNA smear or faint smear on the agarose gel between 200 and 700 bp (step 20).

Potential solution

• •Take 2 μL of DNA sample from step 19 and re-run PCR cycle number optimization step (step 20) using higher PCR cycles. Note that excessive PCR cycles may lead to a significant increase of PCR duplicates in sequencing reads. For deep sequencing, we strongly recommend the optimal PCR cycle for full-scale PCR amplification does not exceed 14.
• •Increase the starting MuSCs cell number in step 1.

Problem 8

The percentage of reported alignments (valid pairs) is too low (step 27).

Potential solution

Improve the proximity ligation efficiency by increasing the amount of T4 DNA ligase and extend the reaction time.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yu Zhao (zhaoyu25@mail.sysu.edu.cn).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Yu Zhao (zhaoyu25@mail.sysu.edu.cn). Questions on using Microcket for processing Hi-C data should be directed to and will be answered by the technical contact, Kun Sun (sunkun@szbl.ac.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate new datasets. The MuSCs Hi-C datasets analyzed in this study are available at Gene Expression Omnibus database: GSE189841. For visualization of Hi-C interaction matrix in Figure 5, we merged all Hi-C replicates generated from FISC or QSC. Instead, Table 1 provides the alignment statistics for a single replicate (QSC: GSM5708470; FISC: GSM5708476), as reported by Microcket.

Acknowledgments

This work was supported by the Shenzhen Science and Technology Program (2023A003 and 20231117190144001 to Y. Zhao), National Natural Science Foundation of China (NSFC; 32270587 and 32100673 to Y. Zhao), and Guangdong Basic and Applied Basic Research Foundation (2021A1515012058 to Y. Zhao and 2023B1515120073 to K.S.).

Author contributions

Y. Zhao conceived and supervised the project. J.K., Y. Zeng, N.L., L.H., and Y. Zhao conducted the related material preparation and biological experiments. K.S. performed bioinformatic analysis. J.K., Y. Zeng, N.L., K.S., and Y. Zhao prepared the first version of the manuscript. H.W., K.S., and Y. Zhao revised the manuscript.

Declaration of interests

The authors declare no competing interests.