GSM9396764: MDA5 siRNA bio rep 3; Homo sapiens; RNA-Seq
RNA was isolated using the Qiagen miRNAeasy mini kit (217084) according to the manufacturer's instructions, followed by DNAse digestion to remove potential genomic DNA contamination. DNAse I treatment was performed at 0.1 U/μl final activity for 15 min at room temperature, followed by the addition of 25 mM EDTA and incubation at 65˚C for 10 min to inactivate DNAse. Total mRNA concentrations were measured using Nanodrop (ThermoFisher Scientific). RNA sequencing was performed by Novogene (Sacramento, CA). mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platforms according to the effective library concentration and the amount of data.