Application of ATAC-Seq for genome-wide analysis of the chromatin state at single myofiber resolution
- Department of Human Genetics, McGill University, Canada
- Lady Davis Institute for Medical Research, Jewish General Hospital, Canada
- McGill Genome Centre, Canada
- Canadian Centre for Computational Genomics, Canada
Figures
Figure 1 with 1 supplement
Schematic of ATAC-seq performed on a single myofiber.
Schematic of the steps and reactions involved in the preparation of sequencing ready libraries of single myofiber DNA for ATAC-Seq. Briefly, myofibers were isolated from the EDL muscle and an …
Figure 1—figure supplement 1
Quality control of ATAC-Seq libraries.
(A) Bioanalyzer profile of an ATAC-Seq library prepared from 5,000 MuSCs. (B) Example bioanalyzer profile of ATAC-Seq library prepared from a single myofiber. (C) Representative picture of a ready …
Figure 2 with 3 supplements
smfATAC-Seq can effectively identify the accessible regions on a single myofiber.
(A) Representative picture of an isolated WT C57BL/6 J uninjured myofiber stained for Hoechst showing the presence and location of myonuclei. Scale bar = 50 µm. (B) Representative picture of an …
Figure 2—figure supplement 1
IGV snapshots of non-myogenic genes.
(A) Platelet and Endothelial Cell Adhesion Molecule 1 (Pecam1) expressed in endothelial cells. (B) Resistin (Retn) as a marker of adipocytes. (C) CD45 expressed in hematopoietic cells. (D) CD90 (Thy1…
Figure 2—figure supplement 2
Correlation analysis between biological replicates of each condition.
(A–C) IGV snapshots of genes expressed in myofibers and MuSCs for all the replicates of each condition that were pooled together for further analysis. DNase-Seq track added to demonstrate …
Figure 2—figure supplement 3
IGV snapshots of Myogenic Regulatory Factors (MRFs).
(A) Myogenic Factor 5 (Myf5). (B) MyoD. (C) Myogenin (Myog). (D) Myogenic factor 6 (Myf6). *ATAC-Seq was performed in biological replicates (n = 3 MuSCs, n = 3 injured myofibers, n = 2 uninjured …
Figure 3 with 3 supplements
Uninjured and injured myofibers and MuSCs display distinct chromatin states.
(A) Heatmap clustering of Pearson correlation coefficients showing the correlation between the replicates of the conditions in the regions defined by the union peakset (merged peaks of all …
Figure 3—figure supplement 1
Correlation analysis between uninjured and injured myofibers only.
(A) Heatmap clustering of Pearson correlation coefficients showing the correlation between the replicates of the injured and uninjured conditions in the regions defined by the union peakset (merged …
Figure 3—figure supplement 2
IGV snapshots of genes expressed in fast and slow muscle fiber types.
(A) Troponin I2 (Tnni2) expressed in fast skeletal muscle fiber. (B) Troponin T3 (Tnnt3) expressed in fast skeletal muscle fiber. (C) Troponin T1 (Tnnt1) expressed in slow skeletal muscle fibers. (D)…
Figure 3—figure supplement 3
Unique peaks between different conditions indicate a distinct chromatin state for each cell type.
(A Peak score distribution (calculated by MACS2 peak calling algorithm)) for each of the different conditions. Peak score = -log10 (FDR). (B) Heatmap showing the read count +/–500 bp of the center …
Figure 4 with 2 supplements
Comparative analysis of chromatin state between uninjured myofibers and MuSCs and between uninjured myofibers and injured myofibers.
(A–C) Gene Ontology (GO Biological Process) analysis of genes associated with ATAC-Seq peaks based on association by proximity using Genomic Regions Enrichment of Annotations Tool (GREAT) (McLean et …
Figure 4—figure supplement 1
Gene Ontology analysis of unique and common peaks between conditions.
(A) Gene Ontology (GO Biological Process) analysis of genes associated with unique peaks present in the uninjured myofiber compared to MuSCs, based on the proximity of the peaks to the genes. (B) GO …
Figure 4—figure supplement 2
Top enriched motifs in the ATAC-Seq peaks of uninjured and injured myofibers.
(A) Top 10 significantly enriched motifs in the peaks that are common between uninjured and injured myofibers overlapping the promoters (±5 kb). (B) Top 10 significantly enriched motifs in the peaks …
Figure 5 with 1 supplement
Identification of cell type specific pathways by global analysis of chromatin accessibility.
(A) Gene Set Enrichment Analysis performed on genes nearest to the differentially accessible regions/peaks for uninjured myofibers compared to injured myofibers. Top 10 enriched pathways are shown …
Figure 5—figure supplement 1
Analysis of Notch and TGFβ signalling pathways reveal differential accessibility between MuSCS and uninjured myofibers, and injured and uninjured myofibers.
(A) Heatmap showing genes involved in the Notch signalling pathway based on read counts of MuSCs, uninjured fibers and injured fibers,±1 kb of the TSS of each gene in the pathway. (B) IGV snapshot …
Figure 6 with 5 supplements
Comparative analysis of chromatin state between MDX and WT myofibers.
(A) Heatmaps showing enrichment at transcription start site (TSS) for the ATAC-Seq libraries of MDX and WT myofibers respectively. (B) Peak annotation pie charts for ATAC-Seq peaks of MDX and WT …
Figure 6—figure supplement 1
Correlation analysis between biological replicates of mdx and WT myofiber ATAC-Seq samples.
(A–I) Scatter plot showing the Pearson correlation between the replicates. (J,K) IGV snapshots of muscle creatine kinase (Ckm) and housekeeping gene Rps2 for all the replicates of each condition …
Figure 6—figure supplement 2
IGV snapshots of myogenic and non-myogenic genes for the quality control of mdx and WT smfATAC-Seq.
(A–F) IGV snapshots of genes known to expressed in muscle fiber displaying accessibility on their respective TSS. (A) The muscle creatine kinase (Ckm). (B) Actin alpha 1 (Acta1). (C) Part of the …
Figure 6—figure supplement 3
Gene Ontology analysis of total mdx and WT peaks.
(A) Gene Ontology (GO Biological Process) analysis of genes associated with all peaks present in the mdx myofiber, based on the proximity of the peaks to the genes. (B) GO term analysis of genes …
Figure 6—figure supplement 4
Correlation analysis between injured, mdx and WT myofibers.
(A) Heatmap clustering of Pearson correlation coefficients showing the correlation between the replicates of the conditions in the regions defined by the union peakset (merged peaks of all …
Figure 6—figure supplement 5
Top enriched motifs in the ATAC-Seq peaks of mdx and WT myofibers.
(A) Top 10 significantly enriched motifs in the peaks that are common between mdx and WT myofibers overlapping the promoters (±5 kb). (B) Top 10 significantly enriched motifs in the peaks that are …
Tables
Table 1
Sequencing read information for smfATAC-Seq and MuSCs ATAC-Seq libraries.
| Library | Number of raw reads | Number of surviving reads | Aligned filtered reads (mm10 reference) | Duplicate reads | Mitochondrial reads | Percentage of mitochondrial reads (%) | Final reads aligned | Number of peaks | Fraction in peaks (FrIP) |
|---|---|---|---|---|---|---|---|---|---|
| Muscle Stem Cells_1 | 175924734 | 113938436 | 103130186 | 47623836 | 529,967 | 0.51 | 54976383 | 65,568 | 0.3642 |
| Muscle Stem Cells_2 | 174965936 | 117357212 | 103570009 | 43672484 | 374,176 | 0.36 | 59523349 | 68,658 | 0.1971 |
| Muscle Stem Cells_3 | 131990380 | 91261584 | 79944121 | 31299456 | 223,540 | 0.28 | 48421125 | 69,573 | 0.1296 |
| Injured_1 | 229935426 | 117212678 | 90040002 | 81024926 | 830,215 | 0.92 | 8184861 | 32,853 | 0.2885 |
| Injured_2 | 194563870 | 129934972 | 98752157 | 88549329 | 1300615 | 1.32 | 8902213 | 28,351 | 0.2863 |
| Injured_3 | 142411536 | 62888552 | 52132455 | 42271079 | 868,808 | 1.67 | 8992568 | 25,002 | 0.2325 |
| Uninjured_1 | 145465410 | 75781456 | 61034569 | 52588315 | 1274332 | 2.09 | 7171922 | 12,276 | 0.2181 |
| Uninjured_2 | 151015852 | 64192706 | 50120282 | 45914841 | 965,037 | 1.93 | 3240404 | 14,742 | 0.3208 |
| MDX_1 | 107540762 | 50979732 | 40485803 | 36205908 | 802,561 | 1.98 | 3477334 | 40,833 | 0.7256 |
| MDX_2 | 103130726 | 54209722 | 46455472 | 37291531 | 1099747 | 2.37 | 8064194 | 39,254 | 0.4932 |
| MDX_3 | 108130662 | 48920904 | 40677359 | 34484003 | 1171316 | 2.88 | 5022040 | 35,691 | 0.5589 |
| WT_1 | 104219578 | 43914902 | 34162142 | 28600498 | 1651199 | 4.83 | 3910445 | 26,873 | 0.7283 |
| WT_2 | 110108692 | 37411936 | 31299222 | 25345321 | 1143317 | 3.65 | 4810584 | 28,430 | 0.64 |
| WT_3 | 183583506 | 72489354 | 56983637 | 49310923 | 1840265 | 3.23 | 5832449 | 39,178 | 0.7611 |
| WT_4 | 86533840 | 36708706 | 28714893 | 25157965 | 1404712 | 4.89 | 2152216 | 21,252 | 0.752 |
Table 2
Percentage of ATAC-Seq peaks that overlap with the TSS±500 bp by at least 1 bp.
| Top 100 genes expressed in whole muscle but not in myofiber | Top 50 genes expressed in whole muscle but not in myofiber | All genes expressed in whole muscle tissue | All genes in the genome | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Number of overlapping peaks | Total number of peaks | % overlapping peaks | Number of overlapping peaks | Total number of peaks | % overlapping peaks | Number of overlapping peaks | Total number of peaks | % overlapping peaks | Number of overlapping peaks | Total number of peaks | % overlapping peaks | |
| Uninjured_Fiber | 12 | 19,704 | 0.0609013 | 3 | 19,704 | 0.0152253 | 7,865 | 19,704 | 39.915753 | 12,995 | 19,704 | 65.951076 |
| Injured_Fiber | 65 | 47,112 | 0.1379691 | 12 | 47,112 | 0.0254712 | 14,259 | 47,112 | 30.266174 | 26,198 | 47,112 | 55.607913 |
| EDL_Whole_Muscle | 198 | 60,719 | 0.3260923 | 65 | 60,719 | 0.1070505 | 18,419 | 60,719 | 30.334821 | 33,048 | 60,719 | 54.427774 |
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Genes identified as being expressed solely in whole muscle but not in myofiber were retrieved from “High-resolution genome-wide expression analysis of single myofibers using SMART-Seq, JBC, Blackburn et al., 2019” and were defined as any gene with an expression of at least 10 RPM in the whole muscle RNA-seq, but 0 RPM in the single myofiber RNA-seq. All genes expressed in whole muscle tissue was defined as any gene that had an RPM value of at least 10 RPM from the whole muscle RNA-seq data by Blackburn et al., 2019 accessible through the GEO accession number GSE138591.
Table 3
Percentage of overlapping peaks between smfATAC-Seq from uninjured myofibers and whole EDL muscle ATAC-Seq.
| Percent overlap (%) | |
|---|---|
| smfATAC-Seq peaks that overlap with EDL-ATAC-Seq by at least 1 bp | 65.9510759 |
| smfATAC-Seq peaks that overlap with EDL-ATAC-Seq by at least 20% | 61.4951279 |
| smfATAC-Seq peaks that overlap with EDL-ATAC-Seq by at least 40% | 52.6136825 |
| smfATAC-Seq peaks that overlap with EDL-ATAC-Seq by at least 60% | 42.1082014 |
| smfATAC-Seq peaks that overlap with EDL-ATAC-Seq by at least 90% | 24.3453106 |
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Whole EDL muscle ATAC-Seq was retrieved from “Dynamic enhancers control skeletal muscle identity and reprogramming, Ramachandran et al., 2019.” This data is accessible through the GEO accession number GSM3981673.
Table 4
Percentage of total peaks found in each genomic feature.
| Muscle stem cells (%) | Injured myofiber (%) | Uninjured myofiber (%) | MDX myofiber (%) | WT myofiber (%) | |
|---|---|---|---|---|---|
| Promoter (±1 kb TSS) | 20.66 | 31.61 | 56.54 | 35.58 | 35.15 |
| Promoter (±1 kb and/or ±2 kb TSS) | 4.81 | 4.84 | 3.45 | 3.78 | 4.53 |
| Promoter ((±2 kb and/or ±3 kb TSS)) | 4.37 | 3.92 | 3.01 | 4.14 | 4.30 |
| 5'UTR | 0.34 | 0.27 | 0.23 | 0.46 | 0.39 |
| 3'UTR | 2.50 | 1.82 | 1.15 | 2.86 | 2.58 |
| First Exon | 1.83 | 1.47 | 1.53 | 1.94 | 1.78 |
| Other Exon | 4.75 | 3.42 | 2.19 | 4.74 | 4.25 |
| First Intron | 11.85 | 10.87 | 7.35 | 10.93 | 10.56 |
| Other Intron | 20.80 | 18.84 | 10.35 | 18.81 | 18.30 |
| Downstream ( ≤ 300 kb) | 1.16 | 1.01 | 0.69 | 1.02 | 0.99 |
| Distal Intergenic | 26.95 | 21.93 | 13.51 | 15.74 | 17.15 |
Table 5
Percentage of differential peaks in each genomic feature.
| Uninjured myofiber vs MuSCs (%) | Uninjured vs injured myofiber (%) | WT vs MDX myofiber (%) | |
|---|---|---|---|
| Promoter (±1 kb TSS) | 43.07 | 25 | 29.92 |
| Promoter (±1 kb and/or ±2 kb TSS) | 3.36 | 7.81 | 3.68 |
| Promoter (±2 kb and/or ±3 kb TSS) | 3.39 | 3.12 | 4.49 |
| 5’UTR | 0.37 | 0.78 | 0.46 |
| 3’UTR | 1.95 | 3.12 | 2.99 |
| First Exon | 2.29 | 3.91 | 1.84 |
| Other Exon | 3.85 | 7.81 | 4.49 |
| First Intron | 9.16 | 13.28 | 14.84 |
| Other Intron | 13.34 | 18.75 | 24.86 |
| Downstream ( ≤ 300 kb) | 0.83 | 0.78 | 0.12 |
| Distal Intergenic | 18.39 | 15.62 | 12.31 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | C57BL/6 J | The Jackson Laboratory | Stock #: 000664 | |
| Genetic reagent (M. musculus) | C57BL/10ScSnJ | The Jackson Laboratory | Stock #: 000476 | |
| Genetic reagent (M. musculus) | C57BL/10ScSn-Dmdmdx/J | The Jackson Laboratory | Stock #: 001801 | |
| Genetic reagent (M. musculus) | Tg(Pax7-EGFE)#Tagb (Pax7-nGFP) | Sambasivan, R. et al. Distinct Regulatory Cascades Govern Extraocular and Pharyngeal Arch Muscle Progenitor Cell Fates. Developmental Cell, (2009). (Sambasivan et al., 2009) | PMID:19531352 | Dr. Shahragim Tajbakhsh (Institut Pasteur) |
| Commercial kit or assay | Tn5 transposase | Illumina | Cat #: 20034197 | |
| Commercial kit or assay | Nextera XT adaptors | Illumina | Cat #: FC-131–1001 | |
| Commercial kit or assay | QIAquick PCR purification kit | Qiagen | Cat #: 28,104 | |
| Chemical compound, drug | Triton X –100 | Sigma-Aldrich | Cat #: T9284 | |
| Chemical compound, drug | Tween-20 | Sigma-Aldrich | Cat #: P1379-1L | |
| Chemical compound, drug | Digitonin | Promega | Cat #: G9441 | |
| Chemical compound, drug | Collagenase D | Roche | Cat #: 11088882001 | 2.4 U/mL |
| Chemical compound, drug | Collagenase | Sigma-Aldrich | Cat #: C0130 | 1000 U/mL |
| Chemical compound, drug | Dispase II | Roche | Cat #: 39307800 | 12 U/mL |
| Chemical compound, drug | Cardiotoxin | Sigma Aldrich | Cat #: 11061-96-4 | |
| Sequence-based reagent | MyoD_L | This paper | PCR primers | TGCTCCTTTG AGACAGCAGA |
| Sequence-based reagent | MyoD_R | This paper | PCR primers | AGTAGGGAA GTGTGCGTGCT |
| Other | Q5 High Fidelity DNA polymerase | New England Biolabs | Cat #: M0491S | For amplification of DNA post Tn5 tagmentation (see Library Preparation) |
| Chemical compound | DAPI stain | Invitrogen | Cat #: D3671 | (5 mg/mL) |
| Other | Ampure XP beads | Beckman | Cat #: A63880 | For library size selection at a concentration of 0.85 x (see Library Preparation) |
| Chemical compound | Hoechst | Molecular Probes | Cat #: H1399 | (5 mg/mL) |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/72792/elife-72792-transrepform1-v2.pdf
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Source data 1
Quality control source data.
(A) Unlabeled agarose gel (1.25%) of MuSC ATAC-Seq sequence ready libraries. (B) Unlabeled agarose gel (1.25%) of uninjured myofiber ATAC-Seq sequence ready library. (C) Labeled agarose gel (1.25%) image of MuSC and uninjured myofiber ATAC-Seq sequence ready libraries. (D) Raw file of bioanalyzer results from single myofiber sequence ready ATAC-Seq libraries.
- https://cdn.elifesciences.org/articles/72792/elife-72792-data1-v2.zip
