Illumina NovaSeq 6000 paired end sequencing; Bulk RNA sequencing of endothelial-derived and non-endothelial-derived BMSCs and OLCs of VE-Cadherin-Cre;LoxP-tdTomato mice
Bulk RNA sequencing of endothelial-derived and non-endothelial-derived BMSCs and OLCs of VE-Cadherin-Cre;LoxP-tdTomato mice
3 or 11 week-old VE-Cadherin-Cre;LoxP-tdTomato mice were euthanized by cervical dislocation. Collagenased bone fraction was obtained from long bones and hips. Cells were stained with the following antibodies: anti-CD45.2 (clone 104, 1:100), anti-Ter119 (clone TER-119, 1:25), anti-CD144 (clone BV13, 1:200), anti-CD51 (clone RMV-7, 1:50), anti-Sca1 (clone D7, 1:50) all from Biolegend, anti-CD140α (clone APA5, 1:100) from eBioscience, and anti-CD31 (clone MEC 13.3, 1:100) from BD Biosciences. Alexa Fluor™ 488-conjugated Streptavidin (1:100, Invitrogen) was used to detect cells marked by biotin anti-CD51 antibody. 7-Amino-Actinomycin D viability dye (7-AAD, 1:100, Beckman Coulter) was used to distinguish between living and dead cells. BMSCs (CD45-TER119-CD31-CD144-SCA-1+ CD51+) and OLCs (CD45-TER119-CD31-CD144-Sca1-CD51+) were directly sorted in Trizol. RNA of sorted cells was extracted using TRIzol according to the manufacturer's instructions for RNA, in combination isolation with GenElute LPA (Sigma-Aldrich).  cDNA was prepared using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech) for Illumina Sequencing.