DOI: 10.5061/dryad.j0zpc86kr
title: Single-cell chromatin accessibility profiling reveals a self-renewing muscle satellite cell state

description: A balance between self-renewal and differentiation is critical for the
 regenerative capacity of tissue-resident stem cells. In skeletal muscle,
 successful regeneration requires the orchestrated activation,
 proliferation, and differentiation of muscle satellite cells (MuSCs) that
 are normally quiescent. A subset of MuSCs undergoes self-renewal to
 replenish the stem cell pool, but the features that identify and define
 self-renewing MuSCs remain to be elucidated. Here, through single-cell
 chromatin accessibility analysis, we reveal the self-renewal versus
 differentiation trajectories of MuSCs over the course of
 regeneration in vivo. We identify Betaglycan as a unique marker of
 self-renewing MuSCs that can be purified and efficiently contributes to
 regeneration after transplantation. We also show that SMAD4 and downstream
 genes are genetically required for self-renewal in vivo by restricting
 differentiation. Our study unveils the identity and mechanisms of
 self-renewing MuSCs while providing a key resource for comprehensive
 analysis of muscle regeneration.
description: ## Single-cell chromatin accessibility profiling reveals a self-renewing
 muscle satellite cell state FigS3M_counting: Number of Fibers per
 microscopy field for SMAD4iKO muscle vs WT. FigS3O_count: Fiber size in
 SMAD4 iKO muscle vs wildtype. The percentage of fibers in various size
 bins is also evaluated. Figure4-raw-images: Immunostaining microscopy
 images of Betaglycan (purple), EdU (green), Pax7 (red) and DAPI (blue) in
 acutely injured muscle at 3 and 4.5 days post injury (4E). 4G: Similar
 immunostaining image for recipient mouse muscle following transplantation
 of lineage traced (GFP) muscle stem cells. Fig_S3-raw-images:
 Immunostaining and H&E images of wildtype and SMAD4 iKO mouse muscle.
 Figure6-raw-images: Immunostaining of cultured SMAD4 iKO and wildtype
 myoblasts with overexpression of Id1, Id2, and Id3 (I), and knockdown of
 Cdkn1c. Figure_5_raw_images: Immunostaining images of WT and SMAD4 iKO
 muscle fibers (E and K) single (E) and repeated (K) acute injuries. In
 vivo immunostaining images of muscle tissue from similar conditions (G and
 M). Fig5H_CTX30D_section_Pax7.xlsx: Quantification of Pax7-expressing
 cells in in Figure_5_raw_images fibers and in vivo after single injury.
 Fig5F.xlsx: Quantification of Pax7-expressing cells in in
 Figure_5_raw_images fibers and in vivo after repeated injury.
 FS3J_adult_section_Pax7.xlsx: Quantification of Pax7-expressing cells per
 100 fibers (defined by Laminin staining in Fig_S3-raw-images) in WT and
 SMAD4 iKO muscle tissue.
 FigS3H_cell_number_on_fiber_at_different_time_points.xlsx: Number of
 Pax7-expressing cells on wildtype and SMAD4 iKO muscle fiber at different
 time points after acute injury. Fig6D.xlsx: Number of satellite cells
 (YFP+) in wildtype and SMAD4 iKO muscle tissue at different time points
 following acute injury.
 Fig6C_cell_number_on_fiber_at_different_time_points.xlsx: Number of
 satellite cells on widltype and SMAD4 iKO muscle fibers at different time
 points following acute injury. Fig5L.xlsx: Pax7+ Satellite cells per
 isolated muscle fiber from wild type (cntrl) and SMAD4 iKO muscle after
 repeated injury. Fig5N_CTX60D_section_Pax7.xlsx: Pax7+ Satellite cells per
 100 in vivo muscle fibers in wild type (cntrl) and SMAD4 iKO muscle after
 repeated injury. Fig4I_quantification.xlsx: Quantification of
 lineage-traced transplanted muscle stem cells that have re-occupied the
 satellite cell niche. Pos and Neg represent Betaglycan-positive muscle
 stem cells and Betaglcan-negative ones. ## Description of the data and
 file structure I have uploaded both raw images (in TIFF or CZI format) and
 image quantification (Microsoft Excel sheets). Most of the images
 represent immuno-staining of muscle tissue based on the questions being
 asked in the article and after different experimental procedures. The gene
 expression data associated with the article has been uploaded on GEO:
 please see the accession number in the article. Excel sheets are
 quantifications of the corresponding microscopy images. Unless indicated,
 numbers are actual counts. In Fig4I_quantification.xlsx, numbers indicate
 mouse (first number, biological replicate), particular muscle section
 (second number), and repeats of each number indicate multiple fields of
 view within that muscle section. In Fig6D.xlsx, numbers that come after
 'WT' or 'KO' are arbitrarily identifying numbers for
 individual mice. In the image files, specific conditions are represented
 by folder names, rather than file names. For example, the
 immono-histological images for transplanted muscle tissue that received
 Betaglycan-positive muscle stem cells are present in the
 Figure4-raw-images folder, Figure 4G sub-folder (because this is the
 specific figure panel in the article that has this image), and in the
 TGFBR3 folder. TGFBR3 is another name for Betaglycan. Also, for image file
 names, C1 to C5, or colors like blue, red, green, represent images from
 various color channels during microscopy. 'Merged' or
 'composite' means the color channels are combined. The rest of
 the texts for particular file names are arbitrary image identifiers e.g.
 the file name 1_red.tif means image 1, red channel. Where letters are
 folder names, they are referring to Figure panels. For example, the E
 folder under Figure_5_raw_images folder contains images for Figure 5E in
 the article. iKO refers to the induced SMAD4 knock-out mutant, and WT
 refers to wild type. Some of the images for the same condition are of
 varying brightness. This is because the pixel range was adjusted to
 improve the identification of nuclei. All the images were followed by
 adequate quantifications gotten by counting fibers and nuclei. Thus, the
 quantification should not be affected by brightness. Also, among the image
 files, some images were reduced in dimension without affecting actual
 pixel content. ## Sharing/Access information Links to other publicly
 accessible locations of the data: Although not included here, the
 associated in-house-generated sequencing data can be downloaded here (raw
 and processed files):
 \*
 Data was derived from the following sources: wildtype and mutant (SMAD4
 iKO) mouse muscle tissue. ## Code/Software Raw microscopy images were
 processed using Image J as described in the article.