RNA sequencing (RNA-SEQ) of WT and LFS patient specific iPSC derived pre-osteoblasts

Purpose: To understand molecular and cellular effects of sFRP2 overexpression in LFS patients Methods: We performed RNA sequencing of 32 samples from WT, sFRP2OE, LFS, and sFRP2 knockdown MSCs or preosteoblasts in four differentiation time points and performed RNAsequencing. Conclusion: molecular and cellular effects of sFRP2 overexpression in LFS patients Overall design: cDNA library preparation and sequencing were performed at Novogen (http://www.novogen.com/home). Briefly, RNA quality was determined with an Agilent Bioanalyzer (RNA integrity number [RIN] > 7.0 for all samples). mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, and double stranded cDNA synthesized using random hexamers, M-MuLV Reverse Transcriptase (RNase H), and DNA Polymerase I. NEBNext Adaptor with hairpin loop structure were ligated to the cDNA in preparation for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments are purified with AMPure XP system (Beckman Coulter, Beverly, USA). Library of 150-200 bp fragments at an effective concentration of > 2nM were loaded onto an Illumina HiSeq 4000 for sequencing