GSM9301779: Trunk neural crest-derived mesenchymal stem cells, 1; Homo sapiens; RNA-Seq
Total RNA was isolated using the RNeasy Micro Kit (Qiagen) and treated with the DNase I kit (Qiagen) to remove any residual genomic DNA following producer's protocols. Then, 10 ng of total RNA was reverse-transcribed into single-stranded cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific) For NGS library construction targeting the Ion AmpliSeq Transcriptome, we utilized the Ion AmpliSeq Transcriptome Human Gene Expression Panel, Chef-Ready Kit (Thermo Fisher Scientific), and the Ion Chef (Thermo Fisher Scientific) instruments, following the manufacturer's protocol. Briefly, a reverse transcription master mix was prepared by combining 5X VILO Reaction Mix and 10X SuperScript III Enzyme Mix. The mix was then aliquoted into reaction plates, with 10 ng of total RNA added to each. The plates were loaded into a thermal cycler and subjected to incubation at 42°C for 30 min, followed by 85°C for 5 min, and then held at 10°C. During library preparation on the Ion Chef instruments, the following consumables and cartridges were loaded: Ion AmpliSeq Chef Solutions DL8 cartridge, Ion AmpliSeq Chef Reagents DL8 cartridge, Ion AmpliSeq Tip Cartridge L8, PCR Frame Seal, IonCode 96 Well PCR Plate, PCR Plate Frame, Empty Tip Cartridge L8, and Enrichment Cartridge. Notably, IonCode 96 Well PCR Plates used for reverse transcription reactions contained an IonCode barcode adapter. Additionally, the tube containing the Ion AmpliSeq Transcriptome Human Gene Expression Panel was inserted into an Ion AmpliSeq Chef Reagents DL8 cartridge. Multiplex PCR was performed in the thermal cycler within the Ion Chef, with 13 cycles of 16-minute annealing/extension reactions. Subsequent steps involved primer digestion with FUPA reagents, ligation of IonCode barcode adapters, library purification using magnetic beads, equalization of library concentrations, and mixing in a single tube