Comparative bulk RNAseq analysis of label-retaining chondrocytes (LRCs) of the growth plate versus their progeny

We revealed the unique molecular signature of slow-cycling chondrocytes of the postnatal growth plate. H2B-GFP(high)tdTomato+ label retaining chondrocytes (LRCs) and H2B-GFP(mid-low)tdTomato+ cells were FACS-isolated from postnatal day 49 (P49) growth plates of Col2a1-tTA; TRE-H2B-GFP; Col2a1-creER; R26R-tdTomato quadruple homozygous mice, with Doxycycline from P21 to P49, and two doses of tamoxifen at 3 and 2 days before analyses. mRNA were amplified by SMARTer mRNA-seq kit (Clonetech/Takara) before deep-sequenced by Illumina HiSeq4000.

本研究揭示了出生后生长板慢循环软骨细胞的独特分子特征。实验所用动物为Col2a1-tTA; TRE-H2B-GFP; Col2a1-creER; R26R-tdTomato四纯合子小鼠，于出生后第21天（P21）至第49天（P49）期间施加多西环素处理，并在分析前3天和2天两次给予他莫昔芬给药。研究人员从该类小鼠的P49生长板中，通过荧光激活细胞分选（FACS）分离得到H2B-GFP(高表达)tdTomato+标记滞留软骨细胞（LRCs）与H2B-GFP(中低表达)tdTomato+细胞。随后采用SMARTer mRNA测序试剂盒（Clonetech/Takara）对提取的mRNA进行扩增，再通过Illumina HiSeq4000平台完成深度测序。