ena-STUDY-SAPIENZA UNIVERSITY OF ROME-14-04-2014-16:40:59:156-78
Discovery of Novel LncRNA species differentially expressed during murine muscle differentiation
By using a strand-specific RNA sequencing approach on growing C2C12 myoblasts and differentiated myotubes (day1, day3 and day5) we identified new murine lncRNAs which expression is dynamically ordered during in vitro differentiation. In order to do that, total RNA from myoblasts/myotubes was isolated with the RNeasy Plus mini kit (Qiagen) according to manufacturer’s instructions and quantified by Nanodrop (Thermo Scientific). RNA-seq libraries were prepared from 200 nanogramms of total RNA using TruSeq™ stranded mRNA Sample Prep Kit (Illumina) and following manufacturer’s instructions. Hence, the transcriptome measurements shown on these tracks were performed on polyA+ selected RNA with the specification of the coding strand. DNA libraries were quantified with KAPA reagent (KAPA SYBR® FAST Universal qPCR Kit, Illumina) and inspected for quality on 2100 Bioanalyzer (Agilent). Multiplexed libraries (10pM) were loaded on the cBot flowcell (TruSeq PE Cluster Kit v2 - cBot–GA) and then sequenced on the GAII (Illumina Inc.) as paired-end 2x86 base reads according to Illumina’s instructions.
By using a strand-specific RNA sequencing approach on growing C2C12 myoblasts and differentiated myotubes (day1, day3 and day5) we identified new murine lncRNAs which expression is dynamically ordered during in vitro differentiation. In order to do that, total RNA from myoblasts/myotubes was isolated with the RNeasy Plus mini kit (Qiagen) according to manufacturer’s instructions and quantified by Nanodrop (Thermo Scientific). RNA-seq libraries were prepared from 200 nanogramms of total RNA using TruSeq™ stranded mRNA Sample Prep Kit (Illumina) and following manufacturer’s instructions. Hence, the transcriptome measurements shown on these tracks were performed on polyA+ selected RNA with the specification of the coding strand. DNA libraries were quantified with KAPA reagent (KAPA SYBR® FAST Universal qPCR Kit, Illumina) and inspected for quality on 2100 Bioanalyzer (Agilent). Multiplexed libraries (10pM) were loaded on the cBot flowcell (TruSeq PE Cluster Kit v2 - cBot–GA) and then sequenced on the GAII (Illumina Inc.) as paired-end 2x86 base reads according to Illumina’s instructions.