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Feb 2022

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ATAC-Seq of a Single Myofiber from Mus musculus
小家鼠单一肌纤维的ATAC-Seq    

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Abstract

Chromatin accessibility is a key determinant of gene expression that can be altered under different physiological and disease conditions. Skeletal muscle is made up of myofibers that are highly plastic and adaptive. Therefore, assessing the genome-wide chromatin state of myofibers under various conditions is very important to gain insight into the epigenetic state of myonuclei. The rigid nature of myofibers, as well as the low number of myonuclei that they contain, have rendered genome-wide studies with myofibers challenging. In recent years, ATAC-Seq from whole muscle and single nucleus ATAC-Seq have been performed. However, these techniques cannot distinguish between different fiber and cell types present in the muscle. In addition, due to the limited depth capacity obtained from single nucleus ATAC-Seq, an extensive comparative analysis cannot be performed. Here, we introduce a protocol where we combine the isolation of a single myofiber with OMNI ATAC-Seq. This protocol allows for genome-wide analysis of accessible chromatin regions of a selected single myofiber at a sufficient depth for comparative analysis under various physiological and disease conditions. This protocol can also allow for a specific myofiber to be selected, such as a regenerating myofiber. In the future, this protocol can help identify global changes in chromatin state under various conditions, as well as between different types of myofibers.


Graphical abstract:




Keywords: Single myofiber (单一的肌纤维), ATAC-Seq (ATAC-Seq), Skeletal Muscle (骨骼肌), Epigenetics (表观遗传学), Chromatin Accessibility (染色质可及性)

Background

Skeletal muscle is the largest tissue in the body and it is primarily composed of myofibers (Buckingham et al., 2003). Myofibers are highly adaptive, and therefore studying changes in myofibers under different stimuli is crucial for understanding how the skeletal muscle adapts and responds to different conditions and diseases (Deschenes, 2004; Wilson et al., 2012). Epigenetics and chromatin accessibility play a key role in the regulation of gene expression and tissue function (Zhu et al., 2018), making it crucial to study chromatin accessibility of different cell types, including myofibers under various stimuli. ATAC-Seq is the commonly used gold-standard method for genome-wide analysis of accessible chromatin regions, and is based on a hyperactive transposase, Tn5 (Buenrostro et al., 2015; Corces et al., 2017). The advancements in sequencing and single cell technologies have allowed for ATAC-Seq to be performed at the whole muscle (Ramachandran et al., 2019) and at the single nucleus levels in recent years (Dos Santos et al., 2020). However, both methods have limitations: whole muscle sequencing does not distinguish between different cell types present in the muscle, and single nucleus ATAC-Seq has lower sequencing depth, restricting its ability for comparative analysis. Therefore, these methods can not assess the chromatin state of only the muscle fibers and can not distinguish different myofiber types. The rigid structure of myofibers (Janssen et al., 2000; Keire et al., 2013) and the low number of myonuclei that a single myofiber contains (200–300 myonuclei) (Neal et al., 2012; Cramer et al., 2020), have made it challenging to perform ATAC-Seq on a single myofiber.


Here, we introduce a method, single myofiber ATAC-Seq (smfATAC-Seq), where we combine the isolation of a single myofiber with OMNI ATAC-Seq (Corces et al., 2017) to analyze the genome-wide accessible chromatin regions of a selected single myofiber without the confounding effects of other cell types present in the muscle. Additionally, this method allows for the selection of a specific myofiber, such as a regenerating myofiber, through the patterning of centrally located myonuclei (Roman and Gomes, 2018). Furthermore, it provides sufficient sequencing depth for peak calling and for comparative analysis of myofibers under various physiological and disease conditions. We have successfully used this method to compare the chromatin accessibility between resting and regenerating myofibers as well as between myofibers isolated from a mouse model of Duchenne Muscular Dystrophy (mdx) and wild-type mice. smfATAC-Seq can allow researchers to assess the chromatin state of a single myofiber and compare the epigenetic changes that occur in myofibers under various biological and disease conditions. Therefore, in the future, smfATAC-Seq might help identify novel mechanistic insights on the role of myonuclei in regulating the plasticity of myofibers under various states.

Materials and Reagents

  1. Materials

    1. 6-well plate (Sarstedt, catalog number: 83.3920)

    2. 1.5 mL microtubes (Sarstedt, catalog number: 72.690.300)

    3. 200 μL strip tubes (Progene, catalog number: 87-C200-8-TB)

    4. Pipette tips (Sarstedt, catalog numbers: 70.3010 [10 μL], 70.3030 [200 μL], 70.3050 [1,000 μL])

    5. Small and Large Glass Pasteur pipettes (VWR, catalog number: 14672-200)

    6. 0.22 μm filter (Ultident, catalog number: 229747)

    7. 1 mL syringe with 26G needle (BD Biosciences, catalog number: 309625)

    8. Microplate (Corning, catalog number: 3676)


  2. Animals

    C57BL/6 mice were acquired from the Jackson Laboratories. The procedure is performed on 4-week-old mice that have gone through intra-muscular cardiotoxin mediated injury on the left hind limb (the procedure has also been successfully performed on 4-week-old uninjured C57BL/6, C57BL/10ScSn-Dmdmdx, and C57BL/10ScSn mice acquired from the Jackson Laboratories). This protocol could be adapted to various strains, but it will be important to adjust the Tn5 dose and incubation time if there is a severe skeletal muscle fiber phenotype.


  3. Reagents

    For Cardiotoxin-mediated injury
    1. Cardiotoxin (CTX) (Sigma, catalog number: 11061-96-4)

    2. Isoflurane (Fresenius Kabi, catalog number: CP0406V2)

    3. PBS (Wisent, catalog number: 311-425-CL)

    4. Carprofen (Zoetis, RIMADYL)

    5. Ethanol (Commercial Alcohols, catalog number: P016EAAN)


    For digestion and isolation of extensor digitorum longus (EDL)

    1. Collagenase from Clostridium histolyctium (Sigma, catalog number: C0130-1G)

    2. PBS (Wisent, catalog number: 311-425-CL)

    3. Horse Serum (Wisent, catalog number: 065-250)

    4. DMEM (Invitrogen, catalog number: 11995073)

    5. Trypsin (Gibco, catalog number: 15090-046)

    6. Isoflurane (Fresenius Kabi, catalog number: CP0406V2)

    7. CO2 (Praxair, catalog number: GP-700500)

    8. Digestion Buffer (see Recipes)

    9. Coating media (see Recipes)


    For selection of injured vs. uninjured myofibers
    1. Hoechst (Molecular Probes, catalog number: H1399)

    2. PBS (Wisent, catalog number: 311-425-CL)


    For lysis and permeabilization of the myofiber
    1. PBS (Wisent, catalog number: 311-425-CL)

    2. Triton X-100 (Sigma, catalog number: T9284)

    3. Permeabilization Buffer (see Recipes)


    For transposition
    1. Tagment DNA Buffer and Tn5 transposase (Illumina, catalog number: 20034197)

    2. Tween-20 (Sigma, catalog number: P1379-1L)

    3. Digitonin (Promega, catalog number: G9441)

    4. PBS (Wisent, catalog number: 311-425-CL)

    5. Nuclease free water

    6. Transposition mixture (see Recipes)


    For DNA purification
    1. QIAquick PCR Purification Kit (Qiagen, catalog number: 28104)

    2. Ethanol (Commercial Alcohols, catalog number: P016EAAN)


    For sequencing-ready library preparation
    1. Q5 High fidelity DNA polymerase (New England Biolabs, catalog number: M0491S)

    2. Deoxynucleotide (dNTP) solution mix (New England Biolabs, catalog number: N0447L)

    3. Nextera XT Index Kit (Illumina, catalog number: FC-131-1001)

    4. Nuclease free water

    5. PCR reaction mixture (see Recipes)


    For library purification and size selection
    1. Ampure XP Beads (Beckman Coulter Life Sciences, catalog number: A63880)

    2. Ethanol (Commercial Alcohols, catalog number: P016EAAN)

    3. Nuclease free water


    For quality control of the sequencing libraries
    1. Quant-IT Picogreen dsDNA Assay kit (Invitrogen, catalog number: P7589)

    2. Tris (Hydroxymethyl) Amino Methane (Multicell, catalog number: 600-125-LG)

    3. Glacial acetic acid (Fisherbrand, catalog number: 351272-212)

    4. Na2EDTA·2H2O (Bioshop, catalog number: EDT 001)

    5. 100 bp DNA ladder (GenedireX, catalog number: DM001-R500)

    6. Agarose (Bioshop, catalog number: AGA001.500)

    7. Orange G (Sigma, catalog number: O3756-25G)

    8. Glycerol (Bioshop, catalog number: GLY001.1)

    9. EDTA (Invitrogen, catalog number: AM9260G)

    10. HCl (Honeywell-Fluka, catalog number: 72787)

    11. GelGreen Nucleic Acid Gel Stain (Biotium, catalog number: 41005)

    12. Primers (Integrated DNA Technologies, standard desalting, 25 nmole)

      VEGFA_TSS_Forward CCGCTGAATAGTCTGCCTTG

      VEGFA_TSS_Reverse GAGAAGCGCAGAGGCTTG

      Chromosome17qE5_Forward TCATCATGTGTCCTGAAGTTGA

      Chromosome17qE5_Reverse GCTTCTCTCCACAGAATTTGC

    13. DNA Loading Dye (see Recipes)

    14. TE Buffer (see Recipes)

    15. TAE Buffer (see Recipes)

Equipment

  1. Pipettes (P20, P200, P1000)

  2. Microscope (Fisher Scientific, inverted microscope, equipped with transmitted light and a 4× objective)

  3. EVOS FLoid Microscope (Life Technologies, catalog number: 4471136)

  4. Dissection tools

    1. Fine point high precision forceps (Fisher Scientific, catalog number: 22-327379)

    2. Sharp-pointed dissecting scissors (Fisher Scientific, catalog number: 08-935)

  5. Cell incubator (Thermo Scientific, Forma Series II Water-Jacketed CO2 Incubator, catalog number: 3110)

  6. Heat block (Fisher Scientific, catalog number: 11-718-2)

  7. Centrifuge (Thermo Scientific, Sorval Legend Micro 21R Microcentrifuge, catalog number: 75002447)

  8. Thermocycler (Bio-Rad, C1000 Touch Thermal Cycler, catalog number: 1851148)

  9. Magnetic rack (Thermo Fisher, DynaMag-2 Magnet, catalog number: 12321D)

  10. Microplate reader (BioTek, synergy4)

  11. Power supply (Bio-Rad, PowerPac, catalog number: 1645050)

  12. Bioanalyzer (Agilent, BioAnalyzer 2100)

  13. Gel Imager (LI-COR, Odyssey FC Imaging System)

Procedure

  1. Intra-muscular injury of mouse Extensor Digitorum Longus (EDL) muscle (optional)

    Note: This step is required if performing ATAC-Seq on regenerating myofibers. If not, you may skip to section B.


    1. Prepare a working solution of 10 µM cardiotoxin (CTX) in PBS; store at -80°C.

    2. Perform a subcutaneous injection of 100 µL per 20 g of mouse body weight of Carprofen (4 mg/mL) and wait 20 min.

      Note: Carprofen is a non-steroidal anti-inflammatory drug (NSAID) used to manage the pain and inflammation that can be associated with the injury.

    3. Anesthetize the mouse using an institutionally approved method. In this protocol, the mouse was anesthetized with 3% isoflurane in an induction chamber.

    4. Spray the hindlimb that is to be injured with 70% ethanol and inject 50 µL of CTX through the Tibialis Anterior (TA) muscle into the EDL using a 1 mL syringe.

    5. Monitor the mouse for 10 min.

    6. Allow the muscle to regenerate for the desired period of time. We performed this on mouse skeletal muscle 7 days post injury; however, different time points after the injury can be used.


  2. Dissection and digestion of Extensor Digitorum Longus (EDL) myofibers from Mus musculus

    1. Prepare a digestion buffer of 1000 U/mL of collagenase from Clostridium histolyctium in unsupplemented DMEM (Recipe 1). Transfer 800 µL of digestion buffer into a 1.5 mL microtube and place it in an incubator at 37°C and 5% CO2.

    2. Coat a 6-well plate with coating media of 10% Horse Serum (HS) in unsupplemented DMEM (Recipe 2). This will prevent the isolated myofibers from sticking to the plate and becoming difficult to handle.

      Notes:

      1. Pure HS or FBS can also be used.

      2. Make sure that the plate is coated for at least 30 min prior to use.

    3. Sacrifice the mouse.

    4. Using a small pair of dissection scissors, remove the skin on the hindlimb and expose the muscles (Video 1).

    5. Dissect the Tibialis Anterior (TA) muscle by cutting the distal tendon, being careful to not cut the adjacent EDL tendon (Video 1).

    6. Peel the TA muscle upwards towards the knee using a pair of forceps. Then cut the TA muscle as close to the knee as possible (Video 1).

    7. Using a pair of forceps, detach the biceps femoris muscle from the knee to expose the proximal tendon of the EDL muscle (Video 1).

    8. Cut the distal EDL tendon and gently pull the EDL upwards towards the knee using a pair of forceps. Ensure that there is a bit of tension in the EDL as you are pulling it up without tugging too hard as that may damage the myofibers (Video 1).

    9. Cut the proximal EDL tendon as close to the knee as possible (Video 1).


      Video 1. Dissection of the EDL muscle.

      The procedure in the video was approved by McGill University Animal Care Committee (UACC) under the protocol #7512.


    10. Place the EDL in the 1.5 mL microtube containing the digestion buffer from step B1. Add trypsin to the digestion buffer at a final concentration of 0.25%.

      Notes:

      1. Trypsin is added to remove the Muscle Stem Cells (MuSCs) associated with the myofibers.

      2. Make sure to add the trypsin at this step and not at Step B1, as Trypsin can digest the collagenase and render it ineffective.

    11. Incubate the EDL muscle with the digestion buffer containing trypsin at 37°C and 5% CO2 for 1 h. Invert the tube periodically for mixing.

    12. During the incubation period, remove the coating media from the 6-well plate and replace it with 2 mL of PBS 1×. Place the plate into the incubator at 37°C and 5% CO2.

      Notes:

      1. The coating media can be kept in the fridge and reused for future myofiber isolations.

      2. Make sure that the plate is placed in the incubator for at least 30 min.

    13. After the 1 h incubation time, transfer the EDL muscle to one of the coated wells of the 6-well plate using a large-bore glass pipette. Coat the large-bore glass pipette with HS prior to use (Figure 1A).

      Note: Coating is very important because myofibers are very sticky in nature.

    14. Gently pipette the EDL muscle up and down with the large-bore glass pipette coated with HS (Figure 1F).

    15. Using a small-bore glass pipette coated with HS, serially transfer a small number of isolated myofibers to the remaining wells of the 6-well plate. This will act as a wash step to remove debris and other cell types.



    Figure 1. Myofiber isolation and selection.

    (A) Typical isolated EDL myofibers in a 6-well plate. (B) Live individual myofiber in a 0.2 mL microtube. (C) Single myofiber in a 0.2 mL microtube post permeabilization. (D) Hoechst staining of an uninjured myofiber. (E) Hoechst staining of a regenerating myofiber, displaying the hallmark of centrally located myonuclei. (F) Glass pipettes used in the procedure. Small-bore glass pipette can be obtained by flame polishing the uncut glass pipette on a Bunsen burner. Large-bore glass pipette can be obtained by cutting a glass pipette and then flame polishing on the Bunsen burner.


  3. Myofiber selection

    1. Add 2 μL of Hoechst (5 mg/mL) into one well of the 6-well plate containing isolated myofibers in 2 mL of PBS 1× (optional).

      Note: If you do not wish to select for a specific myofiber such as a regenerating myofiber, you may skip to step 5.

    2. Place the 6-well plate in a 37°C with 5% CO2 incubator for 5 min.

    3. Remove the 6-well plate from the incubator and place it under the microscope.

    4. If the desired myofiber is an injured myofiber, identify the myofibers with centrally located myonuclei along the length of the myofiber, as opposed to the non-regenerating myofibers with no specific myonuclei patterning (Figure 1D–1E).

      Note: During injury, not all the myofibers undergo regeneration simultaneously or to the same degree. However, regenerating myofibers are characterized by the pattern of centrally located myonuclei. Thus, you may select the regenerating myofiber using this hallmark as a marker.

    5. Coat a small-bore glass pipette with HS.

    6. While visualizing the myofibers under the microscope, select the desired myofiber and transfer it into a 200 μL PCR strip tube using the coated small-bore glass pipette.

    7. Visualize the PCR tube under the microscope to ensure that you have selected and transferred a single myofiber into the tube (Figure 1B).


  4. Myofiber lysis and permeabilization

    1. Using a P200 pipette, remove the excess PBS 1× from the PCR tube under the microscope. Make sure not to remove the myofiber.

    2. Using a P20 pipette, add 20 μL of the permeabilization buffer (Recipe 3) into the tube with the myofiber. Pipette the fiber up and down five times.

    3. Incubate on ice for 15 min (Figure 1C).

    4. Remove the permeabilization buffer with a P20 pipette under the microscope while being careful not to remove the myofiber.

    5. Wash the myofiber by adding 200 μL of PBS 1× using a P200 pipette.

    6. Place the tube on ice for 5 min.

    7. Remove the PBS 1× by using a P200 pipette under the microscope.

    8. Repeat steps 5–7 two times.

    Note: During the 5 min incubation of the washes, you may start the next step for the preparation of the transposition mixture (Recipe 4).


  5. Myofiber transposition by the Tn5 transposase

    1. Prepare the transposition mixture (Recipe 4).

    2. Using a P20 pipette, add 6 μL of the transposition mixture into the tube containing the myofiber. Slowly pipette the myofiber up and down six times.

    3. Place the tube into the heat block set to 37°C for 56 min. Gently shake the tube by flicking it every 5–7 min.

    Note: The Tn5 transposase will cut the accessible chromatin regions and add adaptors simultaneously.


  6. DNA purification

    1. After the transposition, column purify the DNA using the QIAquick PCR Purification Kit according to the manufacturer’s guidelines. Briefly,

      1. Add 70 μL of PB binding buffer into the tube containing the myofiber in the transposition mixture.

      2. Transfer the entire contents of the tube into the column provided by the kit.

      3. Centrifuge the column at 16,000 × g for 1 min.

      4. Discard the flow-through.

      5. Place the column back in the same tube.

      6. Add 700 μL of PE wash buffer into the column.

      7. Centrifuge the column at 16,000 × g for 1 min.

      8. Discard the flow-through. Place the column back in the same tube.

      9. Centrifuge again at 16,000 × g for 1 min.

      10. Discard the flow-through.

      11. Place the column into an empty 1.5 mL microtube.

      12. Add 20 μL of elution buffer into the column. Incubate at room temperature for 5 min.

        Note: You may place the tube at 37°C for 2 min for better elution.

      13. Centrifuge at 16,000 × g for 1 min.

      14. Using a P200 pipette, take out the 20 μL of eluted DNA from the collection tube and add it back into the column for second elution.

        Note: Double elution can help increase the yield.

      15. Elute the DNA again by centrifuging at 16,000 × g for 1 min.

      Note: This is a stopping point. The eluted DNA can be left at 4°C overnight or at -20°C until library preparation.


  7. Preparation of sequencing ready libraries

    1. To amplify and incorporate indices for sequencing, prepare the PCR reaction mix (Recipe 5).

    2. Add 30 μL of PCR reaction mix to the 20 μL of eluted DNA from the previous step for a final volume of 50 μL.

      Note: When preparing the sequencing libraries, make sure that each sample has a different i5 and i7 adaptor combination. In addition, when combining samples in a sequencing lane, make sure that i5 and i7 adaptor combinations for the samples are color balanced for the type of sequencer that is being used (two or four channels). For more information, please visit:

      https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/experiment-design/index-adapters-pooling-guide-1000000041074-05.pdf.

    3. Run the PCR program on the thermocycler using the following steps:

      72°C for 5 min

      98°C for 30 s

      98°C for 10 s

      63°C for 30 s 15 cycles

      72°C for 60 s

      Hold at 4°C

      Note: This is a stopping point. The libraries can be left at 4°C overnight or at -20°C until library purification.


  8. Library purification and size selection

    1. Add AMPure XP beads into the PCR tube at a ratio of 0.85× (v/v). Mix well.

      Note: This ratio is used specifically to remove fragments that are below the size of 200 bp.

    2. Incubate at room temperature for 8 min (Figure 2A).

    3. Place the tube on the magnetic rack. Wait for the solution to become clear (Figure 2B).

    4. Discard the supernatant.

    5. Wash the beads with 200 μL of 80% ethanol.

    6. Remove the ethanol from the tube.

    7. Repeat steps H5–H6. Make sure to remove all the ethanol after the second wash.

    8. Let the beads dry for 2 min.

      Note: Be careful not to over-dry the beads since this leads to lower elution efficiency. However, if the beads do get overdried (a sign of this is the cracking on the beads), do the elution at 37°C for 5–10 min as this can increase the elution efficiency.

    9. Elute the DNA with 15 μL of elution buffer that is provided with the QIAquick PCR Purification Kit. Mix well until beads go into the solution.

    10. Incubate at room temperature for at least 5 min.

      Note: You may place the tubes at 37°C for 2–5 min for better elution.

    11. Place the tube on the magnetic rack. Wait for the solution to become clear.

    12. Keep the supernatant; this is your eluted DNA.

      Note: You may proceed to the quality control steps, or you can store at -20°C until sequencing.



    Figure 2. Size selection and purification of ATAC-Seq libraries by AMPure XP beads.

    (A) AMPure beads in solution. (B) AMPure beads separated from the solution on the magnetic rack.


  9. Quantification and quality control of the ATAC-Seq libraries

    1. Quantify the libraries by using the Quant-IT Picogreen dsDNA Assay kit (Table 1).

      1. Prepare 1× TE buffer from the 20× TE stock solution that is provided with the kit.

      2. Prepare DNA standards by diluting the stock DNA that is provided in 1× TE to the desired concentration.

        Note: You may prepare DNA standards of 10 ng/μL, 5 ng/μL, 2 ng/μL, 1.5 ng/μL, 1.0 ng/μL, 0.75 ng/μL, 0.5 ng/μL, 0.25 ng/μL, 0.1 ng/μL, and 0.05 ng/μL.

      3. Pipette 5 µL of TE 1× buffer into each well of a microplate. Calculate the number of wells needed depending on the number of samples you have. Usually, each standard and sample is run in triplicates.

        Note: Due to the small concentration of DNA following smf-ATAC-Seq , you may run the samples in duplicates instead in order to not waste the samples.

      4. Add 1 μL of the sample and the standards to the corresponding wells containing 5 μL of 1× TE buffer.

      5. Thaw the stock Picogreen dye solution and mix well. Then make a 1:200 dilution of the stock Picogreen in 1× TE buffer.

        Note: Make sure to not expose the Picogreen Dye to direct light for long periods of time.

      6. Pipette 5 μL of the diluted Picogreen solution into each well. Quantify the amount of DNA in each sample by using a microplate reader.

        Note: We usually get a final concentration of 3–7 ng/μL (Table 1).


        Table 1. Representative final concentrations of single myofiber ATAC-Seq libraries

        Sample Concentration (ng/μL)
        Fiber 1 3.59
        Fiber 2 4.61
        Fiber 3 3.76
        Fiber 4 6.97
        Fiber 5 3.39
        Fiber 6 5.66
        Fiber 7 4.08


    1. To verify the library size, run 11 ng of the sample on 1.25% agarose gel with dsGreen (1:10000 dilution) (Figure 3A–3B).

      Notes:

      1. The minimum amount is 11 ng; depending on the amount of DNA, you may run up to 20 ng of the sample on the gel.

      2. Make sure to run only 3 μL of the DNA ladder that is diluted 1:2. This will ensure that the DNA ladder does not wash out the signal from the samples.

    2. To check the enrichment for open regions of chromatin, you may run a qPCR for marker genes in the myofibers (Figure 3C).

      Note: Since myofibers express VEGFA (Lazure et al., 2020), we use the Transcription Start Site (TSS) of VEGFA to check for enrichment. You may use the TSS of other expressed genes in the myofibers. As a negative control, we use a closed region on chromosome 17. We have also included the fold enrichment for the input DNA, which corresponds to the myofibers that were not transposed with the Tn5, called uncut DNA. In a successful ATAC-Seq, you will observe much higher fold enrichments in your sample for your marker gene compared to the enrichment in the Uncut DNA sample.


  10. Run the sequencing libraries on a bioanalyzer (Figure 3D–3F).



    Figure 3. Quality control of smfATAC-Seq libraries.

    (A–B) Representative pictures of smfATAC-seq libraries after size selection, visualized on an agarose gel. (C) qPCR for the TSS of VEGFA compared with a negative control region of Chromosome 17 qE5 for the smfATAC-Seq libraries (n = 5 biological replicates. Error bars = ± SD). (D–F) Examples of bioanalyzer profiles of smfATAC-Seq.

Data analysis

Follow the standard Encode ATAC-Seq pipeline for the analysis. For more information, visit: https://www.encodeproject.org/atac-seq/.

Recipes

  1. Digestion buffer

    1. Dissolve powdered collagenase from Clostridium histolyctium at a final concentration of 1,000 U/mL.

    2. Add trypsin to the buffer at a final concentration of 0.25% once the muscle has been placed in the digestion buffer; this will prevent the trypsin from digesting the collagenase beforehand.

    Note: Always prepare the digestion buffer fresh, immediately before sacrificing the mouse.

  2. Coating media

    Dilute HS into un-supplemented DMEM at a final concentration of 10% (V/V).

  3. Permeabilization buffer

    1. Prepare a stock solution of 10% Triton X-100 in ddH2O.

    2. Prepare a working solution of permeabilization buffer from the 10% stock solution to a final concentration of 0.5% Triton X-100 in ddH2O.

  4. Transposition mixture

    Prepare a transposition mixture for six fibers in a total of 40 μL.

    1. Add 20 μL of Tagment DNA Buffer (TD buffer)

    2. Add 13.3 μL of PBS 1×

    3. Add 4.61 μL of Nuclease free H2O

    4. Add Digitonin for a final concentration of 0.02%

    5. Add Tween-20 for a final concentration of 0.2%

    6. Add 1.39 μL of Tn5

    Note: Add the Tn5 enzyme at the end.

  5. PCR reaction mix (1 reaction, 30 μL)

    10 μL of Q5 Buffer

    10 μL of Q5 enhancer

    1 μL of dNTP’s

    2.5 μL of i7 index

    2.5 μL of i5 index

    3.5 μL of nuclease-free water

    0.5 μL of Q5 High Fidelity DNA polymerase

  6. DNA loading dye

    50% TE Buffer (see Recipe 7)

    50% Glycerol

    Orange G

  7. TE buffer

    1 mL of Tris-HCl pH 8.0, 1M

    200 μL of EDTA pH 8.0, 0.5 M

    Complete to 100 mL with dH2O

  8. TAE buffer

    242 g of Tris base

    57.1 mL of Glacial acetic acid

    37.2 g of anhydrous Na2EDTA

    Complete to 1 L total volume with dH2O

Acknowledgments

This work was funded by a discovery grant from the Natural Sciences and Engineering Research Council (NSERC) to VDS.

This method has been successfully used to analyze the chromatin state of mouse single myofibers under injury and disease conditions (Sahinyan et al., 2022).

Competing interests

The authors declare no competing interests.

Ethics

All procedures that were performed on animals were approved by the McGill University Animal Care Committee (UACC) under the protocol #7512, valid through July/1/2021 – July/1/2022.

References

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  2. Buenrostro, J. D., Wu, B., Chang, H. Y. and Greenleaf, W. J. (2015). ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide. Curr Protoc Mol Biol 109: 21 29 21-21 29 29.
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简介

[摘要]染色质可及性是基因表达的关键决定因素,可在不同生理和疾病条件下改变。骨骼肌由具有高度可塑性和适应性的肌纤维组成。因此,评估各种条件下肌纤维的全基因组染色质状态对于深入了解肌核的表观遗传状态非常重要。肌纤维的刚性以及它们所含的肌核数量较少,使得全基因组研究对肌纤维具有挑战性。近年来,已经进行了来自整个肌肉和单核ATAC-Seq的ATAC-Seq。然而,这些技术无法区分肌肉中存在的不同纤维和细胞类型。此外,由于从单核 ATAC-Seq 获得的深度容量有限,无法进行广泛的比较分析。在这里,我们介绍了一个协议,我们将单个肌纤维的隔离与 OMNI ATAC-Seq 相结合。该协议允许在足够的深度对选定的单个肌纤维的可接近染色质区域进行全基因组分析,以便在各种生理和疾病条件下进行比较分析。该协议还可以允许选择特定的肌纤维,例如再生肌纤维。将来,该协议可以帮助识别各种条件下染色质状态的全局变化,以及不同类型的肌纤维之间的变化。

图形概要:


[背景] 骨骼肌是体内最大的组织,它主要由肌纤维组成(Buckingham等,2003) 。肌纤维具有高度适应性,因此研究不同刺激下肌纤维的变化对于了解骨骼肌如何适应和响应不同的条件和疾病至关重要(Deschenes,2004;Wilson等,2012) 。表观遗传学和染色质可及性在基因表达和组织功能的调节中发挥着关键作用(Zhu et al. , 2018) ,因此研究不同细胞类型的染色质可及性至关重要,包括在各种刺激下的肌纤维。 ATAC-Seq 是对可接近染色质区域进行全基因组分析的常用金标准方法,它基于过度活跃的转座酶 Tn5 (Buenrostro等人,2015;Corces等人,2017) 。近年来,测序和单细胞技术的进步使得 ATAC-Seq 可以在整个肌肉(Ramachandran等人,2019 年)和单核水平(Dos Santos等人,2020 年)进行。然而,这两种方法都有局限性:全肌肉测序不能区分肌肉中存在的不同细胞类型,而单核 ATAC-Seq 的测序深度较低,限制了其比较分析的能力。因此,这些方法不能只评估肌纤维的染色质状态,也不能区分不同的肌纤维类型。肌纤维的刚性结构(Janssen et al. , 2000; Keire et al. , 2013)和单个肌纤维包含的肌核数量少 (200-300 个肌核) ( Neal et al. , 2012; Cramer et al. , 2020) ,这使得在单个肌纤维上执行 ATAC-Seq 变得具有挑战性。
在这里,我们介绍了一种方法,单肌纤维 ATAC-Seq ( smfATAC -Seq),我们将单肌纤维的分离与 OMNI ATAC-Seq (Corces et al. , 2017)结合起来分析全基因组可及的染色质区域一种选定的单一肌纤维,没有肌肉中存在的其他细胞类型的混杂效应。此外,该方法允许通过中央肌核的图案化来选择特定的肌纤维,例如再生肌纤维 (罗曼和戈麦斯,2018 年)。此外,它为峰值调用和各种生理和疾病条件下的肌纤维的比较分析提供了足够的测序深度。我们已经成功地使用这种方法比较了静息和再生肌纤维之间以及从杜氏肌营养不良症 (mdx) 小鼠模型和野生型小鼠中分离的肌纤维之间的染色质可及性。 smfATAC -Seq 可以让研究人员评估单个肌纤维的染色质状态,并比较在各种生物和疾病条件下肌纤维中发生的表观遗传变化。因此,在未来, smfATAC- Seq 可能有助于确定有关肌核在各种状态下调节肌纤维可塑性的作用的新机制见解。

关键字:单一的肌纤维, ATAC-Seq, 骨骼肌, 表观遗传学, 染色质可及性



材料和试剂


A.材料
1.6孔板( Sarstedt ,目录号:83.3920)
2.1.5 mL m微管( Sarstedt ,目录号:72.690.300)
3.200μL条管( Progene ,目录号:87- C200-8 -TB)
4.移液器吸头( Sarstedt ,目录号:70.3010 [10 μL ] ,70.3030 [200 μL ] ,70.3050 [1,000 μL ] )
5.小型和大型玻璃巴斯德移液器(VWR,目录号:14672-200)
6.0.22 μm过滤器(Ultident,目录号:229747)
7.带 26G 针头的 1 mL 注射器(BD Biosciences,目录号:309625)
8.微孔板(Corning,目录号:3676)


B.动物
C57BL/6 小鼠购自杰克逊实验室。该程序在 4周大的左后肢肌肉内心脏毒素介导损伤的小鼠上进行(该程序也已在 4 周大的未受伤 C57BL/6、 C57BL/10ScSn- Dmd上成功进行mdx和从杰克逊实验室获得的C57BL/10ScSn 小鼠)。该协议可以适应各种菌株,但如果存在严重的骨骼肌纤维表型,调整 Tn5 剂量和孵育时间将很重要。


C.试剂
对于心脏毒素介导的损伤
1.心脏毒素(CTX)(Sigma,目录号:11061-96-4)
2.异氟醚(Fresenius Kabi ,目录号:CP0406V2)
3.PBS(Wisent,目录号:311-425-CL)
4.卡洛芬(Zoetis,RIMADYL)
5.乙醇(商业醇,目录号:P016EAAN)


用于消化和分离趾长伸肌 (EDL)
1.来自Clostridium histolyctium的胶原酶(Sigma,目录号:C0130-1G)
2.PBS(Wisent,目录号:311-425-CL)
3.马血清(Wisent,目录号:065-250)
4.DMEM(Invitrogen,目录号:11995073)
5.胰蛋白酶(Gibco,目录号:15090-046)
6.异氟醚(Fresenius Kabi ,目录号:CP0406V2)
7.CO 2 (普莱克斯,目录号:GP-700500)
8.消化缓冲液(见食谱)
9.涂层介质(见配方)


用于选择受伤与未受伤的肌纤维
1.Hoechst(分子探针,目录号:H1399)
2.PBS(Wisent,目录号:311-425-CL)


用于肌纤维的裂解和透化
1.PBS(Wisent,目录号:311-425-CL)
2.Triton X-100(Sigma,目录号:T9284)
3.透化缓冲液(见配方)


换位
1.标记 DNA 缓冲液和 Tn5 转座酶(Illumina,目录号:20034197)
2.Tween-20(Sigma,目录号:P1379-1L)
3.洋地黄皂苷(Promega,目录号:G9441)
4.PBS(Wisent,目录号:311-425-CL)
5.无核酸酶水
6.转座混合物(见配方)


用于 DNA 纯化
1.QIAquick PCR Purification Kit(Qiagen,目录号:28104)
2.乙醇(商业醇,目录号:P016EAAN)


用于准备测序的文库制备
1.Q5高保真DNA聚合酶(New England Biolabs,目录号:M0491S)
2.脱氧核苷酸(dNTP)溶液混合物(New England Biolabs,目录号:N0447L)
3.Nextera XT 索引试剂盒(Illumina,目录号:FC-131-1001)
4.无核酸酶水
5.PCR反应混合物(见配方)


用于文库纯化和大小选择
1.Ampure XP Beads(Beckman Coulter Life Sciences,目录号:A63880)
2.乙醇(商业醇,目录号:P016EAAN)
3.无核酸酶水


用于测序文库的质量控制
1.Quant-IT Picogreen dsDNA Assay 试剂盒(Invitrogen,目录号:P7589)
2.三(羟甲基)氨基甲烷(Multicell,目录号:600-125-LG)
3.冰醋酸( Fisherbrand ,目录号:351272-212)
4.Na 2 EDTA · 2H 2 O( Bioshop ,目录号:EDT 001)
5.100 bp DNA阶梯( GenedireX ,目录号:DM001-R500)
6.琼脂糖( Bioshop ,目录号:AGA001.500)
7.橙色 G(Sigma,目录号:O3756-25G)
8.甘油( Bioshop ,目录号:GLY001.1)
9.EDTA(Invitrogen,目录号:AM9260G)
10.HCl(Honeywell- Fluka ,目录号:72787)
11.GelGreen核酸凝胶染色剂( Biotium ,目录号:41005)
12.引物(集成 DNA 技术,标准脱盐,25 nmole )
VEGFA_TSS_Forward CCGCTGAATAGTCTGCCTTG
VEGFA_TSS_Reverse GAGAAGCGCAGAGGCTTG
Chromosome17qE5_Forward TCATCATGTGTCCTGAAGTTGA
Chromosome17qE5_Reverse GCTTCTCTCCACAGAATTTGC
13.DNA加载染料(见食谱)
14.TE 缓冲液(见配方)
15.TAE 缓冲液(见配方)


设备


1.移液器(P20、P200、P1000)
2.显微镜(Fisher Scientific,倒置显微镜,配备透射光和 4 ×物镜)
3.EVOS FLoid 显微镜(Life Technologies,目录号:4471136)
4.解剖工具
a.细点高精度镊子(Fisher Scientific,目录号:22-327379)
b.尖头解剖剪刀(Fisher Scientific,目录号:08-935)
5.细胞培养箱(Thermo Scientific,Forma Series II Water-Jacketed CO2 Incubator,目录号:3110)
6.加热块(Fisher Scientific,目录号:11-718-2)
7.离心机( Thermo Scientific, Sorval Legend Micro 21R Microcentrifuge,目录号:75002447)
8.热循环仪(Bio-Rad,C1000 Touch 热循环仪,目录号:1851148)
9.磁架(Thermo Fisher,DynaMag-2 Magnet,目录号:12321D)
10.酶标仪 ( BioTek , synergy4)
11.电源(Bio-Rad, PowerPac ,目录号:1645050)
12.生物分析仪(安捷伦, BioAnalyzer 2100)
13.凝胶成像仪(LI-COR,Odyssey FC 成像系统)


程序


A.小鼠指长伸肌 (EDL) 肌肉的肌肉损伤(可选)
注意:如果对再生肌纤维执行 ATAC-Seq,则需要此步骤。如果没有,您可以跳到 B 部分。


1.在 PBS 中制备 10 μM 心脏毒素 (CTX) 的工作溶液;储存在 -80 °C 。
2.每 20 克小鼠体重的卡洛芬(4 毫克/毫升)进行 100 μL 的皮下注射,并等待 20 分钟。
注意:卡洛芬是一种非甾体抗炎药 (NSAID),用于控制与损伤相关的疼痛和炎症。
3.使用机构认可的方法麻醉鼠标。在该协议中,小鼠在感应室中用 3% 异氟醚麻醉。
4.用 70% 乙醇喷洒要受伤的后肢,并使用 1 mL 注射器通过胫骨前 (TA) 肌肉将 50 μL 的 CTX 注入 EDL。
5.监视鼠标 10 分钟。
6.让肌肉在所需的时间内再生。我们在受伤后 7 天对小鼠骨骼肌进行了此操作;但是,可以使用受伤后的不同时间点。


B.Mus musculus指长伸肌 (EDL) 肌纤维的解剖和消化
1.梭菌中制备1000 U/mL胶原酶的消化缓冲液 在未补充的DMEM(配方 1)中。将 800 μL 的消化缓冲液转移到 1.5 mL微管中,并将其置于 37 °C和 5% CO 2的培养箱中。
2.补充的DMEM(配方 2)中涂上 10% 马血清 (HS) 的涂层介质的 6 孔板。这将防止分离的肌纤维粘在板上并变得难以处理。
笔记:
a.也可以使用纯 HS 或 FBS。
b.确保在使用前将板涂上至少 30 分钟。
3.牺牲鼠标。
4.使用一小把解剖剪刀,去除后肢上的皮肤并暴露肌肉(视频 1)。
5.通过切割远端肌腱解剖胫骨前 (TA) 肌肉, 注意不要切割相邻的 EDL 肌腱 (视频 1)。
6.使用一对镊子将 TA 肌肉向上剥离至膝盖。然后尽可能靠近膝盖切割 TA 肌肉(视频 1)。
7.使用一对镊子,将股二头肌从膝盖上分离,以暴露 EDL 肌肉的近端肌腱(视频 1)。
8.切割远端 EDL 肌腱,并使用一对镊子将 EDL 轻轻向上拉向膝盖。确保在拉起 EDL 时有一点张力,不要用力拉得太紧,因为这可能会损坏肌纤维(视频 1)。
9.尽可能靠近膝盖切割近端 EDL 肌腱(视频 1)。




视频 1. EDL 肌肉的解剖。
视频中的程序由麦吉尔大学动物护理委员会 (UACC) 根据协议 #7512 批准。


10.将 EDL 放入包含步骤 B1 中消化缓冲液的1.5 mL 微管中。将胰蛋白酶添加到消化缓冲液中,最终浓度为 0.25%。
笔记:
a.添加胰蛋白酶以去除与肌纤维相关的肌肉干细胞 ( MuSC )。
b.确保在这一步添加胰蛋白酶,而不是在步骤 B1,因为胰蛋白酶可以消化胶原酶并使其失效。
11.用含有胰蛋白酶的消化缓冲液在 37 ° C和 5% CO 2下孵育 EDL 肌肉1 小时。定期倒置试管进行混合。
12.在潜伏期,从 6 孔板中取出涂层介质,并用2 mL 的 PBS 1 ×替换。将盘子放入 37 °C和 5% CO 2的培养箱中。
笔记:
a.涂层介质可以保存在冰箱中,并在未来的肌纤维隔离中重复使用。
b.确保将板放在培养箱中至少 30 分钟。
13.在 1 小时孵育时间后,使用大口径玻璃移液器将 EDL 肌肉转移到 6 孔板的涂层孔之一。使用前用 HS 涂抹大口径玻璃移液器(图 1A)。
注意:涂层非常重要,因为肌纤维本质上非常粘。
14.用涂有 HS 的大口径玻璃吸管上下轻轻吸管 EDL 肌肉(图 1F)。
15.使用涂有 HS 的小口径玻璃移液器,将少量孤立的肌纤维连续转移到 6 孔板的剩余孔中。这将作为清洗步骤以去除碎片和其他细胞类型。








图 1.肌纤维分离和选择。
(A) 6 孔板中典型的隔离 EDL 肌纤维。 (B) 在 0.2 mL 微管中活单个肌纤维。 (C) 0.2 mL 微管透化后的单肌纤维。 (D) 未受伤的肌纤维的赫斯特染色。 (E) 再生肌纤维的赫斯特染色,显示位于中心的肌核的标志。 (F) 过程中使用的玻璃移液器。可以通过在本生灯上对未切割的玻璃移液器进行火焰抛光来获得小口径玻璃移液器。大口径玻璃吸管可以通过切割玻璃吸管,然后在本生灯上进行火焰抛光来获得。


C.肌纤维选择
1.将 2 μL的 Hoechst(5 毫克/毫升)添加到 6 孔板的一个孔中,该孔板在 2 mL 的 PBS 1 × (可选)中含有分离的肌纤维。
注意:如果您不想选择特定的肌纤维,例如再生肌纤维,您可以跳到第 5 步。
2.将 6 孔板放入 37 °C的 5% CO 2培养箱中 5 分钟。
3.从培养箱中取出 6 孔板并将其置于显微镜下。
4.肌纤维的长度识别位于中心的肌核的肌纤维,而不是没有特定肌核图案的非再生肌纤维(图 1D - 1E)。
注意:在受伤期间,并非所有的肌纤维都同时或相同程度地进行再生。然而,再生肌纤维的特点是位于中央的肌核模式。因此,您可以使用此标志作为标记来选择再生肌纤维。
5.用 HS 涂抹小口径玻璃移液器。
6.在显微镜下可视化肌纤维时,选择所需的肌纤维并使用涂层小口径玻璃移液器将其转移到 200 μL PCR 条管中。
7.在显微镜下可视化 PCR 管,以确保您已选择并将单个肌纤维转移到管中(图 1B)。


D.肌纤维溶解和透化
1.使用 P200 移液器,在显微镜下从 PCR 管中取出多余的 PBS 1 × 。确保不要移除肌纤维。
2.使用 P20 移液器,加入 20 μL透化缓冲液(配方 3)与肌纤维一起进入管中。上下移取纤维五次。
3.在冰上孵育 15 分钟(图 1C)。
4.在显微镜下用 P20 移液器去除透化缓冲液,同时注意不要去除肌纤维。
5.使用 P200 移液器加入 200 μL的 PBS 1 ×清洗肌纤维。
6.将试管置于冰上 5 分钟。
7.在显微镜下使用 P200 移液器去除 PBS 1 × 。
8.重复步骤 5 – 7 两次。
注意:在洗涤液的 5 分钟孵育期间,您可以开始下一步准备转座混合物(配方 4)。


E.Tn5转座酶的肌纤维转座
1.准备转置混合物(配方 4)。
2.使用 P20 移液器,将 6 μL的转位混合物添加到含有肌纤维的管中。慢慢地上下移取肌纤维六次。
3.将管子放入设置为 37 °C 的热块中 56 分钟。每5-7 分钟轻弹一次,轻轻摇动试管。 
注意:Tn5 转座酶将切割可接近的染色质区域并同时添加接头。


F.DNA纯化
1.转座后,根据制造商的指南,使用QIAquick PCR Purification Kit 对 DNA 进行柱纯化。简要地,
a.将 70 μL的 PB 结合缓冲液添加到转位混合物中含有肌纤维的管中。
b.将管中的全部内容物转移到试剂盒提供的柱中。
c.16,000 × g离心柱 1分钟。
d.丢弃流通。
e.将色谱柱放回同一管中。
f.加入 700 μL的 PE 洗涤缓冲液。
g.16,000 × g离心柱 1分钟。
h.丢弃流通。将色谱柱放回同一管中。
i.16,000 × g离心 1分钟。
j.丢弃流通。
k.将色谱柱放入空的 1.5 mL 微管中。
l.加入 20 μL的洗脱缓冲液。在室温下孵育 5 分钟。
注意:您可以将管子在 37 °C 下放置 2 分钟以获得更好的洗脱效果。
m.16,000 × g离心机 1分钟。
n.使用 P200 移液器,从收集管中取出 20 μL洗脱的 DNA,并将其添加回柱中进行第二次洗脱。
注意:双重洗脱有助于提高产量。
o.16,000 × g离心再次洗脱 DNA 1分钟。
注意:这是一个停止点。洗脱的 DNA 可以在 4 °C 下放置过夜或在 -20°C 下放置直到文库制备。


G.测序就绪文库的制备
1.要放大和合并测序指标,请准备 PCR 反应混合物(配方 5)。
2.将 30 μL的 PCR 反应混合物添加到上一步中洗脱的 20 μL的 DNA 中,最终体积为 50 μL 。
注意:准备测序文库时,请确保每个样本都有不同的 i5 和 i7 适配器组合。此外,在测序通道中组合样本时,请确保样本的 i5 和 i7 适配器组合针对正在使用的测序仪类型(两个或四个通道)进行颜色平衡。欲了解更多信息,请访问:
https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/experiment-design/index-adapters-pooling-guide-1000000041074-05.pdf 
3.使用以下步骤在热循环仪上运行 PCR 程序:
72 °C 5 分钟
98°C 30 秒
98°C 10 秒
63°C 30 秒 15 个循环
72°C 60 秒
保持在 4°C
注意:这是一个停止点。文库可在 4°C 或 -20°C 下放置过夜,直至文库纯化。


H.文库纯化和大小选择
1.× (v/v)的比例将AMPure XP 珠子添加到 PCR 管中。搅拌均匀。
注意:此比率专门用于去除大小低于 200 bp 的片段。
2.在室温下孵育 8 分钟(图 2A)。
3.将试管放在磁架上。等待解决方案变得清晰(图 2B)。
4.弃去上清液。
5.μL的 80% 乙醇清洗珠子。
6.从管中取出乙醇。
7.重复步骤H5 - H6 。确保在第二次洗涤后除去所有乙醇。
8.让珠子干燥 2 分钟。
注意:注意不要过度干燥磁珠,因为这会导致洗脱效率降低。但是,如果珠子确实过度干燥(这表明珠子开裂),请在 37 °C 下洗脱 5 – 10 分钟,因为这可以提高洗脱效率。
9.μL洗脱 DNA 与QIAquick PCR Purification Kit一起提供的洗脱缓冲液。充分混合,直到珠子进入溶液。
10.在室温下孵育至少 5 分钟。
注意:您可以将管子在 37 °C 下放置 2 – 5 分钟以获得更好的洗脱效果。
11.将试管放在磁架上。等待溶液变得清晰。
12.保留上清液;这是您洗脱的 DNA。
注意:您可以继续质量控制步骤,或者您可以在 -20°C 下储存直到测序。




AMPure XP 磁珠对 ATAC-Seq 文库的大小选择和纯化。
(A)溶液中的AMPure珠子。 (B) AMPure珠子从磁性架上的溶液中分离出来。


I.ATAC-Seq 文库的量化和质量控制
1.使用Quant-IT Picogreen dsDNA 检测试剂盒对库进行量化(表 1)。
a.从套件随附的 20 × TE 储备溶液中制备 1 × TE 缓冲液。
b.× TE中提供的储备 DNA 稀释至所需浓度来制备 DNA 标准品。
注意:您可以制备 10 ng/ μL 、5 ng/ μL 、2 ng/ μL 、1.5 ng/ μL 、1.0 ng/ μL 、0.75 ng/ μL 、0.5 ng/ μL 、0.25 ng/ μL 、0.1 ng的 DNA 标准品/ μL和0.05 ng/ μL 。
c.将 5 μL 的 TE 1 ×缓冲液移入微孔板的每个孔中。根据您拥有的样品数量计算所需的孔数。通常,每个标准和样品一式三份运行。
注意:由于smf -ATAC-Seq 后的 DNA 浓度很小,您可以重复运行样本,以免浪费样本。
d.将 1 μL的样品和标准添加到含有 5 μL 1 × TE 缓冲液的相应孔中。
e.解冻Picogreen染料溶液并充分混合。然后在 1 × TE 缓冲液中对Picogreen股票进行 1:200 稀释。
注意:确保不要将Picogreen染料长时间暴露在直射光下。
f.5 μL稀释的Picogreen溶液移液到每个孔中。使用酶标仪量化每个样本中的 DNA 量。
注意:我们通常得到的最终浓度为 3 – 7 ng/ μL (表 1)。


表 1. 单个肌纤维 ATAC-Seq 文库的代表性最终浓度


2.要验证库大小,请使用dsGreen (1:10000 稀释)在 1.25% 琼脂糖凝胶上运行 11 ng 样品(图 3A - 3B)。
笔记:
a.最小量为 11 ng;根据 DNA 的量,您最多可以在凝胶上运行 20 ng 的样品。
b.确保只运行 3 μL 1:2 稀释的DNA 梯。这将确保 DNA 梯不会洗掉样本中的信号。
3.要检查染色质开放区域的富集情况,您可以对肌纤维中的标记基因运行 qPCR(图 3C)。
注意:由于肌纤维表达 VEGFA( Lazure等人,2020),我们使用 VEGFA 的转录起始位点 (TSS) 来检查富集。您可以使用肌纤维中其他表达基因的 TSS。作为阴性对照,我们使用 17 号染色体上的封闭区域。我们还包括输入 DNA 的折叠富集,它对应于未用 Tn5 转座的肌纤维,称为未切割 DNA。在成功的 ATAC-Seq 中,您将观察到与未切割 DNA 样品中的富集相比,您的样品中标记基因的富集倍数要高得多。


J.在生物分析仪上运行测序库(图 3D - 3F)。






图 3. smfATAC -Seq 文库的质量控制。 
(A - B)大小选择后smfATAC- seq 库的代表性图片,在琼脂糖凝胶上可视化。 (C) VEGFA的 TSS 的 qPCR与smfATAC- Seq 库的染色体 17 qE5 的阴性对照区域相比(n = 5 个生物复制。误差线 = ± SD)。 (D – F) smfATAC -Seq 的生物分析仪配置文件示例。


数据分析


按照标准编码 ATAC-Seq 管道进行分析。欲了解更多信息,请访问: https://www.encodeproject.org/atac-seq/ 。


食谱


1.消化缓冲液
a.溶解梭菌中的粉状胶原酶,最终浓度为 1 , 000 U/ mL。 
b.将肌肉置于消化缓冲液中后,将胰蛋白酶以 0.25% 的终浓度添加到缓冲液中;这将阻止胰蛋白酶预先消化胶原酶。
注意:在牺牲鼠标之前,始终准备新鲜的消化缓冲液。
2.涂层介质
将 HS 稀释到未补充的 DMEM 中,最终浓度为 10% (V/V)。
3.透化缓冲液
a.2 O中制备 10% Triton X-100 的库存溶液。
b.2 O中制备从 10% 库存溶液到最终浓度为 0.5% Triton X-100 的通透缓冲液的工作溶液。
4.转座混合物
为总共 40 μL的六根纤维准备转置混合物。
a.添加 20 μL Tagment DNA 缓冲液(TD 缓冲液)
b.添加 13.3 μL的 PBS 1 ×
c.添加 4.61 μL无核酸酶 H 2 O
d.添加洋地黄皂苷,最终浓度为 0.02%
e.添加 Tween-20 使最终浓度为 0.2%
f.添加 1.39 μL的 Tn5
注意:在末尾添加 Tn5 酶。
5.PCR 反应混合物(1 个反应,30 μL )
10 μL Q5 Buffer
10 μ L Q5 增强剂
1 μL dNTP's
2.5 μL i7 指数
2.5 μL i5 指数
3.5 μL无核酸酶水
0.5 μL Q5 高保真 DNA 聚合酶
6.DNA上样染料
50% TE 缓冲液(见配方 7)
50% 甘油
橙色 G
7.TE缓冲液
1 mL Tris-HCl pH 8.0, 1M
200 μL EDTA pH 8.0, 0.5 M
用 dH 2 O完成至 100 mL
8.TAE 缓冲液
Tris基础242g
57.1 毫升冰醋酸
37.2 克无水 Na 2 EDTA
2 O完成至 1 L 的总体积


致谢


这项工作由自然科学和工程研究委员会 (NSERC) 向 VDS 提供的一项发现资助资助。
该方法已成功用于分析损伤和疾病条件下小鼠单肌纤维的染色质状态( Sahinyan等,2022) 。


利益争夺


作者声明没有竞争利益。


伦理


对动物进行的所有程序均由麦吉尔大学动物护理委员会 (UACC) 根据协议 #7512 批准,有效期至 2021 年 7 月/1 日至 2022 年 7 月 1 日。


参考


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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Sahinyan et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Sahinyan, K., Blackburn, D. M. and Soleimani, V. D. (2022). ATAC-Seq of a Single Myofiber from Mus musculus. Bio-protocol 12(12): e4452. DOI: 10.21769/BioProtoc.4452.
  2. Sahinyan, K., Blackburn D. M. and Soleimani V. D. (2022). Application of ATAC-Seq for genome-wide analysis of the chromatin state at single myofiber resolution. eLife 11: e72792.
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