Single-cell transcriptomic analysis of the identity and function of fibro/adipogenic progenitors in healthy and dystrophic muscle

. 2023 Jul 23;26(8):107479. doi: 10.1016/j.isci.2023.107479

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Experimental models: Organisms/strains

BLA/J	The Jackson Labs	RRID:IMSR_JAX:012767
C57BL/6J	The Jackson Labs	RRID:IMSR_JAX:000664
D2-mdx	The Jackson Labs	RRID:IMSR_JAX:013141
C57BL/10J	The Jackson Labs	RRID:IMSR_JAX:000665

Antibodies

PDGFRα - Rabbit anti-mouse (1:250)	Cell Signaling	RRID:AB_2162345; 3174S (D1E1E)
PDGFRα - Goat anti-mouse (1:50)	RND Systems	RRID:AB_2236897; AF1062
PDGFRα-APC	BioLegend	RRID:AB_2043970; 135908
ADAMTS5 - Rabbit anti-mouse (1:100)	Abcam	RRID:AB_2222327; AB41037
DPEP1 - Rabbit anti-human/mouse (1:100)	Abcam	RRID:AB_11133237; AB121308
C3 - Rat anti-mouse (1:250)	Novus Biologicals	NB200-540
IL-33 – Rat anti-mouse (1:250)	RND Systems	MAB3626
Alexa Fluor 488 - Chicken anti-rabbit (1:500)	Thermo Fisher	RRID:AB_2535859; A-21441
Alexa Fluor 594 - Goat anti-rabbit (1:500)	Thermo Fisher	AB_2534079: A-11012
Alexa Fluor 594 - Chicken anti-goat (1:500)	Thermo Fisher	AB_2535871; A-21468
Alexa Fluor 647 - Chicken anti-rabbit (1:500)	Thermo Fisher	AB_2535861; A-21443

Critical commercial assays

RNAscope Multiplex Fluorescent Reagent Kit V2	Acdbio	323100
TaqMan Fast Advanced Master Mix	ThermoFisher	4444557
Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1, 4 rxns	10X Genomics	1000128
Chromium Next GEM Chip G Single cell kit, 16 rxns	10X Genomics	1000127
Single Index Kit T Set A, 96 rxns	10X Genomics	1000213

Chemicals, peptides, and recombinant proteins

Pdgfra	Acdbio	480661-C2
Dpep1	Acdbio	480831-C3
C3	Acdbio	417841-C3
Il33	Acdbio	400591-C3
Adamts5	Acdbio	427621
Opal 690 (1:750)	Akoya Biosciences	FP1497001KT
Opal 570 (1:750)	Akoya Biosciences	FP1488001KT
Opal 520 (1:750)	Akoya Biosciences	FP1487001KT
SuperScript IV VILO Master Mix	Thermo Fisher	11756050
C3 - Mm01232779_m1 (FAM-MGB)	Thermo Fisher	4331182
Cd248 - Mm00547485_s1 (FAM-MGB)	Thermo Fisher	4331182
Dpep1 - Mm00514592_m1 (FAM-MGB)	Thermo Fisher	4331182
Hprt1 (control) - Mm00446968_m1 (VIC-MGB)	Thermo Fisher	4448490
Il33 - Mm00505403_m1 (FAM-MGB)	Thermo Fisher	4331182
Ly6a (SCA-1) - Mm04337234_m1 (FAM-MGB)	Thermo Fisher	4331182
Pdgfra - Mm00440701_m1 (FAM-MGB)	Thermo Fisher	4331182
Collagenase II (2.5U/mL)	Thermo Fisher	17101015
Collagenase D (1.5U/mL)	Sigma Aldrich	COLLD-RO
Dispase II (2.4U/mL)	Sigma Aldrich	D4693
High Sensitivity DNA Kit (D5000)	Agilent	5067-4626
Qubit dsDNA HS Assay Kit	Thermo Fisher	Q328541
TURBO DNAse	Thermo Fisher	AM2238
ProLong™ Gold Antifade Mountant with DNA Stains DAPI	Thermo Fisher	P36935
Wheatgerm Agglutinin (1:1000)	Thermo Fisher	W6748
7AAD (1:200)	BioLegend	420404

Deposited data

scRNA-seq data of WT and BLA/J FAPs generated for this manuscript (FASTQ & MTX files)	This manuscript	GEO:
Scripts and Seurat objects (“faps_mesc_analysis.zip”)	This manuscript	https://doi.org/10.5281/zenodo.8102775
Tabula Muris single cell RNA-seq data set (20 tissues including limb muscle)	Tabula Muris consortium	tabula-muris.ds.czbiohub.org/
Giordani et al mouse limb muscle scRNA-seq data set	Giordani et al.¹²	GEO:GSE110878
“scMuscle_mm10_slim_v1-1.RData” integrated mouse skeletal muscle sc/snRNA-seq data set	McKellar et al.³⁶	github.com/mckellardw/scMuscle
“Mouse_SS_Fibro.RDS”, steady state mouse fibroblast dataset	Buechler et al.²⁴	Fibroxplorer.com

Software and algorithms

CellSens Software (v3.1.1)	Olympus	olympus-lifescience.com/en/software/cellsens/
FIJI/ImageJ (v2.1.0/1.53c)	FIJI Team	fiji.sc
Prism 9.3.1	Graphpad	graphpad.com/scientific-software/prism
Cell Ranger 3.0.0	10X Genomics	support.10xgenomics.com/single-cell-gene-expression/software/overview/welcome
GSEA	Subramanian et al.¹⁷	gsea-msigdb.org
Cytoscape	Shannon et al.¹⁹	cytoscape.org
EnrichmentMap pipeline collection (EnrichmentMap, AutoAnnotate, WordCloud, clusterMaker2)	Reimand et al.¹⁸	apps.cytoscape.org/apps/enrichmentmappipelinecollection
R Programming Language	The R Foundation	r-project.org
Seurat (version 2-4, see scripts)	Satija Lab⁵²^,⁵³^,⁵⁴	satijalab.org/seurat
data.table	Dowle and Srinivasan⁵⁵	github.com/Rdatatable/data.table
ggplot2	Wickham H⁵⁶	ggplot2.tidyverse.org/
reshape2	Wickham H⁵⁶	github.com/hadley/reshape
Dplyr	Wickham et al.⁵⁷	dplyr.tidyverse.org
Tibble	Muller and Wickham⁵⁸	tibble.tidyverse.org
Plyr	Wickham H⁵⁶	plyr.had.co.nz/
harmony	Korsunsky et al.⁵⁹
RColorBrewer	Brewer, CA	ColorBrewer.org
patchwork	Pedersen, TL	github.com/thomasp85/patchwork/
DropletUtils	Marioni Lab	github.com/MarioniLab/DropletUtils/
sctransform	Hafemeister et al.⁶⁰	github.com/satijalab/sctransform
cluster	Maechler et al.⁶¹	CRAN.R-project.org/package=cluster
Pheatmap	Kolde et al.⁶²	github.com/raivokolde/pheatmap
cowplot	Wilke Lab	wilkelab.org/cowplot/