For RNA extraction of mSSCs, 500 to 1,000 cells were FAC-sorted directly into 1 mL TRIzol LS (Invitrogen, Cat#10296028). For bulk tissues, bones were flash frozen in liquid nitrogen, crushed into powder, and added directly into 1 mL TRIzol. RNA was isolated with the RNeasy Micro Kit (Qiagen, Cat#74004) and twice amplified with an Arcturus™ RiboAmp™ PLUS Kit (Applied Biosystems™, Cat#KIT0521).
Label
streptavidin
Label protocol
RNA was streptavidin-labeled as recommended by Affymetrix protocol.
Hybridization protocol
RNA was fragmented and hybridized to an Affymetrix Mouse 430 2.0 array (Applied Biosystems™, Cat#900495).
Scan protocol
Arrays were scanned with a Gene Chip Scanner 3000 (Affymetrix) running the GCOS 1.1.1 software.
Data processing
For mSSCs profiled from P3, Ad, and Ad/MF joints, the corresponding CEL files were normalized by MAS5 using the affy v1.66.0 R package, mapped to NCBI Entrez gene identifiers using a custom chip definition file for Mouse Genome 430 2.0 microarrays (‘mouse4302mmentrezgcdf’), and converted to Mouse Genome Informatics (MGI) gene symbols using the org.Mm.eg.db v3.11.1 R package. Gene expression values were log2-normalized prior to downstream analysis. For bulk tissues and mSSCs from Ad/MF joints treated with control (PBS), BMP2, and VEGFR1, the corresponding CEL files were normalized using the Gene Expression Commons platform (https://gexc.riken.jp) and. Briefly, gene expression profiles were normalized against a common reference of >11,939 Affymetrix Mouse Genome 430 2.0 microarrays. Each gene’s expression was then transformed to percentile ranks (range: –100% to +100%) based on its relative value to the reference. Two matrices consisting of (1) percentile ranks or relative gene expression activity from GEXC for bulk tissues and mSSCs from Ad/MF joints treated with control (PBS), BMP2, and VEGFR1 and (2) custom-cdf- and MAS5-normalized expression for mSSCs profiled from P3, Ad, and Ad/MF joints.
