Establishment of patient-derived bone organoids to recapitulate and dissect Hurler syndrome's skeletal disease
Establishment of patient-derived bone organoids to recapitulate and dissect Hurler syndrome's skeletal disease
To induce cartilaginous differentiation of MPS IH and HD BMSCs the micro-mass culture system was used as previously described (Serafini et al 2014). Pellets were snap frozen Pellets were thawed and homogenized by Mikro-Dismembrator U (Gottingen). Total RNA was extracted using TRIzol reagent (Invitrogen), according to manufacturer&#39;s protocol. Whole transcriptome RNA-seq analyses have been performed by Next Generation Sequencing (NGS), using the Universal RNAseq kit (Nugen, Tecan).The Universal RNA-Seq is an end-to-end solution for strand-specific RNA-Seq library construction used 250 ng nanogram amounts of total RNA obtained from gut sections. The workflow consists of double-stranded cDNA generation using a mixture of random priming, optional fragmentation of double-stranded cDNA, end repair, adaptor ligation, strand selection, targeted transcript depletion with AnyDeplete for globin genes, and PCR amplification to produce the final library. The yields of final libraries were assessed by Qubit 4.0 fluorimeter and their sizing by Agilent Bioanalyzer.