GSM7677195: miRNA vEDS fibroblast P5; Homo sapiens; miRNA-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 QIAxcel (QIAGEN) and the Qubit fluorometer (Thermofisher Scientific), respectively. To ensure the accuracy and reproducibility of the data, a biological replicate (BR) was prepared for each patient and control sample, resulting in a total of 72 miRNAs libraries. Reverse transcription and library preparation were achieved following the standard protocol of the QIAseq miRNA Library kit (QIAGEN). All procedures were carried out in accordance with the manufacturer's instructions. This method incorporates unique molecular indexes during cDNA synthesis, which uniquely tag each strand before amplification, resulting in a more accurate reflection of endogenous miRNA levels by controlling for library amplification bias (Wong et al., 2019). Libraries were quantified with the QIAxcel (QIAGEN) diluted to 100 pM and pooled equally, with 6 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.