GSM6732656: Control fibroblast C25; Homo sapiens; RNA-Seq
Total RNA was purified from patient and control cells grown for 72 hours at 70-80% of confluency using the miRNeasy kit (Qiagen). Sample quality and quantity were evaluated with the Agilent 2100 Bioanalyzer (Agilent Technology) and the Qubit fluorometer (Thermofisher Scientific), respectively. To improve statistical power, RNA samples from all experimental groups were sequenced twice as biological replicates (BR) (at the 2nd and 4th in vitro passage, respectively) plus 20 technical replicates (TR) for control fibroblasts and 10 for both hEDS and HSD cells. Reverse transcription and library preparation were achieved following the standard protocol of the Ion Ampliseq Transcriptome Human Gene Expression Kit (Thermofisher Scientific), which targets by multiplexed PCR 18,574 protein-coding and 2,228 non-coding RNAs (mRNAs/ncRNAs). Libraries were quantified with the Ion qPCR Library Quantification Kit, diluted to 100 pM and pooled equally, with 8 samples per pool, followed by emulsion PCR and enrichment with the Ion OneTouch2 with ES system. Libraries were then loaded onto an Ion 540 chip and sequenced on the Ion S5 instrument. RNA-seq, targeted transcriptome sequencing