Supplementary Figures Legends.

Supplementary Figure 1. Validation of myonuclear labeling by H2B-GFP. a, Efficient

labeling of myonclei by H2B-GFP in uninjured and regenerating muscles 7 days after cardiotoxin

(CTX) injury (7 d.p.i.). Note the centralized nuclei in 7 d.p.i. Scale bar, 30 µm. b, Efficient labeling

of myonuclei in isolated single EDL fiber. DAPI was used as a counterstain to identify all nuclei.

Scale bar, 100 µm. c, Specificity of H2B-GFP expression in myofibers. Double

immunoflourescence of GFP and markers of endothelia, Schwann cells, macrophages and muscle

stem cells demonstrates the absence of H2B-GFP in these cell types. Scale bar, 50 µm. d, Sorting

strategy of myonuclei isolation. After gating based on FSC/SSC (P1), doublets were removed (P2

and P3), DAPI positive nuclei were gated (P4), and DAPI/GFP double positive nuclei were sorted

(P5). Percentages of gated nuclei (relative to total events) at each step are indicated. DAPI/GFP

images before and after sorting are shown on the right. Scale bar, 30 µm.

Supplementary Figure 2. Quality control plots for the datasets used in this study. a, Number

of detected UMIs per nucleus when considering both exonic and intronic reads (left) or only exonic

reads (right). b, Number of detected per nucleus when considering genes detected in at least

5 nuclei. c, Mitochondrial reads percentage thresholds for each batch. For all box plots, the center lines indicate median, the end points of the boxes indicate low and high quartiles, and the whiskers indicate minimum and maximum values. Also see Supplementary Table 5 for additional quality control information.

Supplementary Figure 3. Myh2 can be co-expressed with Myh1, but not with Myh4. Single molecule FISH of Myh1, Myh2 and Myh4 in uninjured TA muscle. The probe against Myh2 detected pre-mRNA and therefore appears as puncta; the probes against Myh1 and Myh4 detected processed mRNA located mainly in the cytoplasm. (Most right) Overlay of Myh2 and Myh4 does not show co-expression of these two . (Middle) Myh1 and Myh2 can be co-expressed in the same fiber, which are indicated in arrows. Scale bar, 30 µm. Expression patterns were validated in more than 3 individuals.

Supplementary Figure 4. Genes with dynamic expression profile during regeneration. a,

Violin plots showing the expression level of indicated genes in uninjured, 7 d.p.i. and 14 d.p.i. muscles. b, RT-qPCR analysis of indicated genes using isolated GFP+ nuclei at different time points. Error bars indicate S.E.M. Two-tailed unpaired student’s t-test (n=3 for each time point).

*, p < 0.05. **, p < 0.01. ***, p < 0.001. c, Single molecule FISH of indicated genes at different time points. Scale bar, 50 µm. Expression patterns were validated in 2 or more individuals.

Supplementary Figure 5. Expression of Tigd4, a marker of MTJ-A, in uninjured TA muscle.

Tile scan image of single molecule FISH against Tigd4. DAPI was used as a counterstain to identify all nuclei. The insets indicate the MTJ area that is magnified in the right panels. Expression patterns were validated in more than 3 individuals. Scale bars, 200 µm (for the entire tile scan) and

100 µm (for the insets).

Supplementary Figure 6. FISH validation of the expression of marker genes identified in the

MTJ-B cluster. Conventional FISH (Col6a3 or Pdgfrb) and GFP immunofluorescence at the MTJ

in uninjured (top two rows) or 14 d.p.i. TA muscle expressing H2B-GFP in myonuclei (middle

two rows). T indicates tendon and M myofiber; arrowheads point to myonuclei that co-express marker genes and GFP. The bottom two rows show regions distal to the MTJ in uninjured muscle where expression of marker genes is also observed in GFP-negative nuclei. Note that due to high temperatures used during the conventional FISH (but not single molecule FISH) procedure, GFP signals are dampened and not observed in all myonuclei. Expression patterns were validated in 2 or more individuals. Scale bar, 50 µm.

Supplementary Figure 7. Quantification of repeat elements in the snRNAseq dataset.

Unsupervised hierarchical clustering of the repeat element expression profile across nuclear populations. The most frequently expressed 16 repeat elements are presented. Note the strong enrichment of srpRNA in Gssos2+ nuclei.

Supplementary Figure 8. Rian and Gssos2 are expressed in distinct nuclear populations. a,

Single molecule FISH of Rian and Gssos2 in uninjured TA muscle shows that Rian expressing

nuclei (asterisk) and Gssos2 expressing nuclei (arrows) are distinct. b, Single molecule FISH of

Rian/Ufsp1 and Gssos2/Ufsp1 shows that Rian and Gssos2 expressing nuclei (arrows) are distinct

from Ufsp1 expressing nuclei. Expression patterns were validated in 2 or more individuals. Scale

bars, 30 µm.

Supplementary Figure 9. Re-clustering of the bulk myonuclei identified in Figure 1. Labeling

of different time points (left) and expression profile of Ttn (right).

Supplementary Figure 10. Further characterization of nuclear subtypes in Mdx muscle. a,

Expression of Ttn and . Note that Dystrophin expression is homogenous across all

nuclear populations. b, MA plot of enriched genes in the ‘dying fiber’ cluster in Figure 4a; Exp.,

expression. c, Co-expression of indicated ncRNAs in a subset of myonuclei. Scale bar, 30 µm. d,

Fibers containing such nuclei are infiltrated with H2B-GFP negative cells in c. Scale bar, 30 µm.

e, Location of MTJ-A in the Mdx UMAP map. Scales indicate signature scores of MTJ-A

nuclei. f, Single molecule FISH of NMJ markers (Ufsp1 and Prkar1a) or MTJ-A markers (Tigd4

and Col22a1) in control and Mdx muscle. Note that the strict co-expression of Prkar1a and Ufsp1

is weakened in Mdx muscle. In contrast, Tigd4 and Col22a1 are strongly co-expressed in Mdx

muscle. Scale bar, 50 µm.

Supplementary Figure 11. Myonuclei expressing a repair gene signature in Mdx and

Dysferlinopathy muscles. a, Single molecule FISH images for Flnc/Xirp1 in wild-type mouse or

muscle of a healthy human donor. These FISH experiments were conducted at the same time as

those shown in Figures 4f and 4g. b, Co-expression of indicated marker genes in muscle of Mdx

mice. c, Co-expression of Flnc and Xirp1 in the muscle of mice carrying a mis-sense mutation

(L1360P) in the Dysferlin gene. d, Co-expression of FLNC and XIRP1 in a muscle biopsy of a human patient carrying a homozygous mutation in the DYSFERLIN gene (DYSF c.4872delG); nuclei co-expressing these genes were also identified in a second patient. Scale bars, 50 µm.

Expression patterns in mouse tissues were validated in 2 or more individuals.

Supplementary Figure 12. Validation of nuclear compartments in the muscle spindle. a, Two different protocols were used to isolate spindle myonuclei, and this does not bias their distribution in the UMAP plot. See methods for further details of these protocols. b, Expression of indicated marker genes by single molecule FISH in cross-sections of TA muscle expressing H2B-GFP after

Calb1-Ires-Cre recombination. Scale bars, 10 µm. Expression patterns were validated in 2 or more individuals.

Supplementary Figure 13. Loss of Ebf1 does not affect expression of marker genes in NMJ,

MTJ-A and Rian+ nuclei. Single molecule FISH of indicated marker genes in TA muscle of control or Ebf1 mutant mice. Expression patterns were validated in 3 individuals. Scale bar, 30

µm.

Supplementary Figure 14. Uncropped Western blot images for figure 6c.

Supplementary Fig. 1

aLaminin GFP b Isolated EDL fiber Control H2B-GFP No injury No 7 d.p.i. 7 DAPI GFP

c Endothelia Schwann cells Macrophages Stem cells

Cd31 Egr2 F4/80 Pax7 GFP or DAPI ColIV

d 250 250 P4 (41.3%) DAPI GFP 200 P1 (50.4%) 200 Before 150 150 100 100

⁵ P5 (22.2%) 50 SSC-A (x1000) 50 10 SSC-A(x1000) ⁴

50 100 150 200 250 -10³ 0 10³ 10⁴ 10⁵ 10

FSC-A (x1000) DAPI-A ³ 10 DAPI-A After

0 After 250 ³ P2 (48.1%) 250 P3 (45.4%) -10 200 200 -10³ 0 10³ 10⁴ 10⁵ 150 150 GFP-A 100 100 50 SSC-W (x1000) 50 SSC-W (x1000) 50 100 150 200 250 50 100 150 200 250 FSC-H (x1000) FSC-H (x1000) Supplementary Fig. 2

a 50000 Exonic and intronic reads 15000 Exonic reads only (used here)

40000 10000 30000

20000 5000 10000 nUMI per nucleus per nUMI 0 0

b 6000 Detected at least in 5 nuclei c 2.5

2.0 4000 1.5

1.0 2000 0.5 nGene per nucleus per nGene Mitochondria reads % reads Mitochondria 0 0.0 Supplementary Fig. 3

Myh1 Myh2 Myh4 (uninjured) Supplementary Fig. 4

a Uninjured 7 d.p.i. 14 d.p.i. Arrdc2 Pdk4 Pfkfb3 Gpt2 Ptp4a3 Pik3ip1 Igf2bp3 4 4 3 5 4 3 3 4 3 3 2 3 2 2 3 2 2 2 1 2 1 1 1 1 1 1 0 0 0 0 0 0 0 Smox Glul Slc26a2 Cish F830016B08Rik Mettl21c Ddit4l 4 4 3 3 4 3 3 4 3 3 2 2 2 2 2 2 2 1 1 1 1 1 1 0 0 0 0 0 0 0

Expression level Expression Bhlhe40 Apoe Gm13340Casq2 Nmrk2 Acta1 Fabp3 4 4 5 3 3 3 3 3 3 4 2 2 3 2 2 2 2 2 1 1 1 1 1 1 1 0 0 0 0 0 0 0

b Uninjured 7 d.p.i. 14 d.p.i. Arrdc2 Pfkfb3 Smox Gpt2 Cish Ddit4l Casq2 Acta1 * * ** ** * * ** 1.5 1.5 1.5 1.5 * 10 6 8 15 * * * * *** 8 6 1.0 1.0 1.0 1.0 4 10 6 4 4 0.5 0.5 0.5 0.5 2 5 2 2 0.0 0.0 0.0 0.0 0 0 0 0 Relative expression Relative

c Arrdc2 Smox Pdk4 Slc26a2 Mettl21c Nmrk2 Uninjured 14 d.p.i. 7 7 d.p.i. d.p.i. 14

ColVI DAPI Supplementary Fig. 5

DAPI

Tigd4 Supplementary Fig. 6

MTJ-B GFP DAPI M M M M T Pdgfrb

(MTJ) M M

Uninjured M T Col6a3

M M T Pdgfrb (MTJ)

14 d.p.i. 14 T M Col6a3 Pdgrfb Uninjured (distal toMTJ) (distal Col6a3 Supplementary Fig. 7

srpRNA rRNA scRNA Simple repeat tRNA RNA SINE snRNA Satellite Low complexity Alu B2 8 ERVK LINE LTR

0 RC Clusters -A B + - ulk 2+ TJ B an+12 sos2+MTJ NMJ s M Ri uz Bcl G S Supplementary Fig. 8

a Gssos2 Rian GFP ColIV (uninjured)

* *

b Rian Ufsp1 GFP (uninjured) Gssos2 Ufsp1 GFP Supplementary Fig. 9

Re-clustering of bulk myonuclei

Uninjured Ttn 7 d.p.i. 14 d.p.i.

Perimysium Perimysium 0 8 Supplementary Fig. 10

a b Markers of dying fibers Ttn Dystrophin Gm10717 Gm10801 1.5 Gm10719 Gm21738 Gm10800 1.0 Gm10722 Gm11168 Gm26870 0.5

0.0

log2 (exp ncRNA rest) (exp / log2 -0.5 0 8 0 6 0 2 4 6 Average expression

c GFP Gm10800 Gm10801 d GFP DAPI

e MTJ-A signature score f NMJ MTJ-A Control

0 0.5 Mdx

Prkar1a Ufsp1 Tigd4 Col22a1 DAPI DAPI Supplementary Fig. 11

a Wild-type mouse Healthy human Flnc Xirp1 GFP Klhl40 Egr1 GFP FLNC XIRP1 DAPI ColIV

b Mdx GFP ColIV Flnc Xirp1

Klhl40 Egr1

c Dysferlin L1360P mouse DAPI ColIV Flnc Xirp1

d Dysferlinopathy patient DAPI ColIV FLNC XIRP1 Supplementary Fig. 12

a b Sens. spdNMJ spdMTJ

Calcrl Ufsp1 Ebf1 GFP

Protocol 1 Protocol 2 DAPI ColIV Supplementary Fig. 13

Control Ebf1 Mut Ufsp1 (NMJ) DAPI Prkar1a1 Col22a1 (MTJ-A) DAPI Tigd4 Ttn (Rian+) Rian DAPI Supplementary Fig. 14

Strip from Ebf1, Blot: Ebf1 Re-blot: β- Blot: ColVI

180kD 180kD 180kD 130kD 130kD 130kD ColVI 100kD 100kD 100kD 70kD 70kD 70kD 55kD 55kD 55kD 40kD 40kD 35kD 35kD 35kD 25kD 25kD 25kD 15kD 15kD 15kD 10kD 10kD 10kD