Expression profiling of LINC01638 interference in hMSC-hTERT20 cells
We identified LINC01638 as a lncRNA that is highly expressed in proliferating MSCs and decreases in expression during osteogenesis. We demonstrate that knockdown of LINC01638 in hMSC-hTERT20 cells by CRISPRi during osteogenic differentiation causes an decrease in alkaline phosphatase (ALP) activity and ALPL gene expression. Altered expression of several extracellular matrix proteins was noted. We identified LINC01638 as a nuclear lncRNA that binds to chromatin and where it regulates gene expression.  These results reveal that LINC01638 may act as a negative regulator of osteoblast differentiation, thereby providing a novel mechanism of regulatory control of osteogenesis. Overall design: RNA-Seq: Human immortalized MSCs (hMSC-hTERT20) were infected with pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-GFP to induce LINC01638 or LINC01114 knockdown. Cells were then induced to osteogenesis using 280 µM ascorbic acid, 10 mM beta-glycerophosphate, 10nM dexamethasone. RNA from cells were harvested at defined time points (0, 7, 14,21 days) in trizol. RNA was hen processed into individually barcoded libraries and sequenced.